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UNIVERSIDADE ESTADUAL DO CEARÁ

PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA

FACULDADE DE VETERINÁRIA

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS VETERINÁRIAS

JOHANNA LEIVA REVILLA

Teste de toxicidade da fração da Auxemma oncocalyx e onconcalyxona A sobre o

desenvolvimento folicular e embrionário in vitro

FORTALEZA – CEARÁ

2016

JOHANNA LEIVA REVILLA

TESTE DE TOXICIDADE DA FRAÇÃO DA AUXEMMA ONCOCALYX E

ONCONCALYXONA A SOBRE O DESENVOLVIMENTO FOLICULAR E

EMBRIONÁRIO IN VITRO

Tese apresentada ao Curso de Doutorado em

Ciências Veterinárias do programa de Pós-

graduação em Ciências Veterinárias da Faculdade

de Veterinária da Universidade Estadual do

Ceará, como requisito parcial à obtenção do título

de doutor em Ciências Veterinárias. Área de

Concentração: Reprodução e Sanidade Animal.

Orientador: Prof. Dr. José Ricardo de Figueiredo

FORTALEZA – CEARÁ

2016

Dedico,

A minha família, Jorge e Patricia, porque

eles me deram o seu amor e apoio

incondicional e constante durante toda a

minha vida.

Aos meus amigos, por seu inabalável

apoio, amizade e amor.

AGRADECIMENTOS

À Universidade Estadual do Ceará (UECE), ao Programa de Pós-Graduação em Ciências

Veterinárias (PPGCV), aos professores e aos funcionários por esses anos colaborando

com minha formação profissional.

Ao Laboratório de Manipulação de Oócitos inclusos em Folículos Pré-Antrais

(LAMOFOPA) da UECE, por dar-me todo o suporte para a realização dessa tese.

À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelo

incentivo financeiro, através da bolsa de doutorado concedida.

Ao meu orientador, professor Dr. José Ricardo de Figueiredo, pela oportunidade de

ingressar em sua equipe, pela confiança depositada, por todos os ensinamentos, por me

orientar na execução desta tese e por me incentivar cada vez mais no exercício das minhas

capacidades, e por acreditar no meu trabalho.

À Profa. Dra. Ana Paula Ribeiro Rodrigues, pela confiança concedida e orientação

durante meu doutorado, que através do seu exemplo profissional foi de grande

importância no meu crescimento professional.

Aos membros da banca, Profa. Dra. Juliana Jales de Hollanda Celestino, Prof. Dr. Dárcio

Ítalo Alves Teixeira, Dr. Luis Alberto Vieira, Prof. Dr. José Roberto Viana da Silva e a

Dra. Ticiana Franco Pereira da Silva pelas correções desta tese, contribuindo para torna-

la ainda melhor.

À Carolina Maside Mielgo e Luis Alberto Vieira, por me co-orientar e pelo exemplo como

profissionais e amigos, sempre com a maior e melhor vontade de me ajudar em tudo.

Obrigada por ser parte da minha família, vocês são pessoas incríveis!

À Profa. Dra. Juliana Jales de Hollanda Celestino, pela constante assessoria, ajuda e

correções de todos meus artigos.

À minhas bruxas queridas, Anna Clara Accioly Ferreira (Chats) e Denise Damasceno

Guerreiro (Denaise), obrigada por tanta amizade, força, motivação e tantos momentos

cheios de alegrias, eu vou levar vocês sempre no meu coração.

A Jesús de los Reyes Cadenas Moreno, por ter sido meu irmão e braço direito durante

meus experimentos, por ter tido tanta paciência comigo, por todas as receitas de cozinha,

muito obrigada. Você sempre vai ser uma parte da minha família.

À Giovanna e à minha Família brasileira, os Quintino, Camila, Michelle, Lourdes e

Antonio Airton. Obrigada por ter me recebido de braços abertos e como se fosse uma

filha. Serei eternamente grata por ter ganho uma família cheia de tanto amor.

À Laritza Ferreira Lima e Simone Vieira Castro, pela ajuda e orientação durante meu

início no LAMOFOPA.

A Renato Felix da Silva, pelos milhões de capilares e pela amizade. Obrigada filho.

À Francisco Leo Nascimento de Aguiar (Pequeño Leo), pelas palavras de motivação,

incentivo, ajuda, amizade e pela grata companhia durante as viagens a Mossoró.

A Victor Macêdo Paes (Estrela) e Hudson Henrique Vieira Correia (Huds), pelos debates

no horário de almoço, o companheirismo, a ajuda e amizade.

Prof. Claudio Cabral e Benner Geraldo Alves, pelas mil e uma análises de estatística.

Obrigada pela paciência.

A toda equipe do LAMOFOPA que me auxiliou de alguma forma e as pessoas que

fizeram meus dias mais felizes: Rita Kelly, Lidiane Sales, Seu João, César Camelo, Gildas

Mbemya Tetaping, Juliana Zani, Naiza Arcângela Ribeiro de Sá, Nathalie Jiatsa, Yago

Pinto, Andreza de Sá Nunes, Gerlane Modesto, Gabriel da Silva, Leticia Ferreira, Deysi

Dipaz Berrocal, Diego Montano, Kayse Najara, Luana Gaudencio, Luciana Mascena

Silva, Marcela Pinheiro Paz, Geovania Canafístula, Rebeca Rocha, Kele Amaral Alves,

Jamily Bruno, Valdevane Araujo e Carlos Lobo.

E, por fim um agradecimento especial à minha família, Jorge e Patricia, vocês são minha

razão de viver, sem vocês eu não teria conseguido. Muito obrigada por todo o amor e

apoio que sempre me deram. Amo muito vocês!

RESUMO

O objetivo foi avaliar o efeito da Auxemma oncocalyx (A. oncocalyx) e seu composto

ativo oncocalyxona A (onco A) sobre os folículos pré-antrais, a maturação oocitária e a

qualidade embrionária in vitro. Para a Fase I, fragmentos ovarianos caprinos foram

cultivados por 1-7 dias nos tratamentos: controle cultivado sozinho ou suplementado com

DMSO, BMP-15, doxorubicina (DXR), A. oncocalyx (1.2, 12 ou 34 µg/mL) ou onco A

(1, 10 ou 30 µg/mL). Foram avaliados: sobrevivência, crescimento, apoptose e

proliferação. Na fase II, folículos secundários isolados caprinos foram cultivados nos

grupos; controle não-cultivado, controle cultivado, DMSO, DXR, A. oncocalyx ou onco

A. Além disso, complexo culmulus oócito (CCOs) caprinos foram maturados in vitro

(MIV) nos grupos: controle-cultivados sozinho; ou suplementado com DMSO; DXR; A.

oncocalyx ou onco A, e foram avaliados a morfologia, sobrevivência, apoptose e

configuração da cromatina. Finalmente, para a fase III, CCOs porcinos foram MIV na

presença de DXR; A. oncocalyx ou onco A e a competência oocitária foi avaliada

(experimento 1). Também, embriões porcinos foram cultivados in vitro nos mesmos

tratamentos e o desenvolvimento embrionário foi avaliado (experimento 2). Na fase I, a

DXR reduziu o percentual de viabilidade folicular sendo que para a A. oncocalyx e onco

A esta redução ocorreu de forma concentração-dependente (P <0,05). DXR, A. oncocalyx

1.2 e onco A 1 aumentaram (P <0,05) a apoptose. A DXR diminuiu (P<0,05) a

proliferação celular. Porém, na fase II, houve uma redução no percentual de folículos

intactos, formação antro, taxa de crescimento e proliferação celular (P <0,05) comparado

ao controle. A DXR apresentou indicadores de apoptose maiores (P <0,05). Após a MIV

de CCOs caprinos, DXR, A. oncocalyx e onco A aumentaram (P <0,05) os oócitos

anormais e diminuíram a viabilidade em comparação com o controle (P <0,05).

Finalmente, na fase III, no experimento 1, a DXR, A. oncocalyx e onco A reduziram (P

<0,05) a viabilidade oocitária e a eficiência da MIV (P <0,05). Após a FIV, todas as

drogas reduziram (P <0,05) a eficiência da FIV e o percentual de embriões clivados, no

entanto, apenas a DXR diminuiu o percentual de blastocistos. No experimento 2, a DXR

e A. oncocalyx diminuíram (P <0,05) o percentual de embriões clivados, mas não teve

nenhum efeito sobre a formação de blastocisto. Em conclusão, A. oncocalyx e onco A

afetam a foliculogênese in vitro em caprinos de forma concentração-dependente. A Onco

A tem um efeito menos prejudicial do que a DXR na sobrevivência de folículos pré-

antrais. Também, A. oncocalyx e onco A não apresentam um efeito tóxico sobre folículos

secundários isolados caprinos nas taxas de maturação de CCOs. Porém, estas substâncias

afetam negativamente a viabilidade oocitária. Além disso, em suínos, a adição de DXR

durante MIV ou cultivo embrionário in vitro afeta negativamente a eficiência da FIV e a

taxa de clivagem. Além disso, a exposição de CCOs suínos à DXR, apenas durante a

MIV, foi mais prejudicial à manutenção da viabilidade oocitária e à formação de

blastocistos, quando comparado à A. oncocalyx e onco A.

Palavras-chave: Auxemma oncocalyx. Oncocalyxona A. Cultivo in vitro. Maturação in

vitro. Embriões.

ABSTRACT

The goal was to evaluate the effect of Auxemma oncocalyx (A. oncocalyx) and its active

compound oncocalyxone A (onco A) on the in vitro culture of preantral follicles, oocyte

maturation and embryo quality. For phase I, caprine ovarian fragments were cultured for

1 and 7 days in different conditions: cultured control alone or supplemented with DMSO,

BMP-15, doxorubicin (DXR), A. oncocalyx (1.2, 12 or 34 µg/ml) or onco A (1, 10 or 30

µg/ml). The following endpoints were evaluated: survival, growth, apoptosis and

proliferation. For phase II, isolated secondary caprine follicles were cultured in groups;

non-cultured control, control group, DMSO, DXR, A. oncocalyx or onco A. Additionally,

caprine cumulus-oocyte-complex (COCs) were in vitro maturated (IVM) in the groups:

control alone or supplemented with DMSO; DXR; A. oncocalyx or onco A. Morphology,

survival, apoptosis and chromatin configuration were assessed. Finally, for phase III,

porcine COCs were IVM in the presence of DXR; A. oncocalyx or onco A and oocyte

competence was analyzed (experiment 1). Also, porcine embryos were in vitro cultured

in the same treatments and embryo development was evaluated (experiment 2). In phase

I, A. oncocalyx and onco A, in a concentration-dependent manner, and DXR decreased

(P<0.05) the percentage of morphologically normal follicles. DXR, A. oncocalyx 1.2 and

onco A 1 increased (P<0.05) the percentage of apoptosis. DXR decreased (P<0.05) the

cellular proliferation. On the other hand, in phase II, DXR showed a reduction in the

percentage of intact follicles, antrum formation, growth rate and proliferation (P < 0.05)

compared to control. DXR showed higher apoptosis indicators (P < 0.05). After IVM of

caprine COCs, DXR, A. oncocalyx and onco A treatments had a greater percentage (P <

0.05) of abnormal oocytes and a lower percentage of viable oocytes as compared with the

control (P < 0.05). Finally, in the phase III, in experiment 1; DXR, A. oncocalyx and onco

A reduced (P<0.05) oocyte viability and IVM efficiency. After IVF, all the drugs reduced

(P<0.05) the IVF efficiency and percentage of cleaved embryos, nevertheless, only DXR

decreased the percentage of blastocyst. In experiment 2; DXR and A. oncocalyx decreased

(P<0.05) the percentage of cleaved embryo, but had no effect on blastocyst formation. In

conclusion, A. oncocalyx and onco A affected in vitro caprine folliculogenesis in a

concentration-dependent manner. Onco A has a less harmful effect than DXR on goat

preantral follicle survival. Furthermore, A. oncocalyx and onco A do not exhibit a toxic

effect on caprine isolated secondary follicles and on maturation rates of COCs recovered

from caprine antral follicles. However, these substances negatively affected the oocyte

viability. Moreover, in the porcine model, the addition of DXR during IVM or IVC

negatively affected the IVF efficiency and cleavage rate. Additionally, the exposure of

porcine COCs to DXR only during IVM was more detrimental to oocyte viability and

blastocyst formation than A. oncocalyx and onco A.

Keywords: Auxemma oncocalyx. Oncocalyxone A. In vitro culture. In vitro maturation.

Embryos.

LISTA DE FIGURAS

FRAÇÃO DE AUXEMMA ONCOCALYX E ONCOCALYXONA A

AFETAM A SOBREVIVÊNCIA IN VITRO E O DESENVOLVIMENTO DE

FOLÍCULOS PRÉ-ANTRAIS CAPRINOS INCLUSOS EM TECIDO

CORTICAL OVARIANO

Figure 1 - TUNEL assay of caprine preantral follicles before (non-cultured

control) and after in vitro culture for 7 days in α-MEM+ alone or α-

MEM+ supplemented with DMSO, BMP-15, DXR, A. oncocalyx

(1.2 µg/mL) or onco A (1 µg/mL) …………………………………. 77

Figure 2 - AgNOR silver staining of caprine preantral follicles before (non-

cultured control) and after in vitro culture for 7 days in α-MEM+

alone or α-MEM+ supplemented with DMSO, BMP-15, DXR, A.

oncocalyx (1.2 µg/mL) or onco A (1 µg/mL) ……………………… 77

Figure 3 - PCNA test of caprine preantral follicles before (non-cultured

control) and after in vitro culture for 7 days in α-MEM+ alone or α-

MEM+ supplemented with DMSO, BMP-15, DXR, A. oncocalyx

(1.2 µg/mL) or onco A (1 µg/mL) ………………………………… 78

EFEITO DA TOXICIDADE DA FRAÇÃO DA AUXEMMA ONCOCALYX E

DO PRINCÍPIO ATIVO ONCOCALYXONA A NO CULTIVO IN VITRO

DE FOLÍCULOS SECUNDÁRIOS E NA MATURAÇÃO IN VITRO DE

OÓCITOS CAPRINOS

Figure 1 - Isolated secondary follicles before (a) and after 7 days of culture in

α-MEM+ alone (cultured-control) (b) or supplemented with DMSO

(c), DXR (d), A. oncocalyx (e) and onco A (f). Oocytes after in vitro

maturation in TCM199+ (control) (g) or supplemented with DMSO

(h), DXR (i), A. oncocalyx (j) or onco A (k) ………………………. 104

Figure 2 - Relative mean (± SEM) of BAX:BCL2 mRNA ratio in cultured

isolated secondary follicles for 7 days in α-MEM+ alone (cultured-

control) or supplemented with DMSO, DXR, A. oncocalyx and

onco A. Different letters denote significant differences (P < 0.05) .. 104

AUXEMMA ONCOCALYX E SEU COMPOSTO ATIVO

ONCOCALYXONA A PREJUDICAM A COMPETÊNCIA DE

DESENVOLVIMENTO OOCITÁRIO IN VITRO EM SUÍNOS, MAS SÃO

MENOS PREJUDICIAIS DO QUE A DOXORRUBICINA

Figure 1 - Experimental design and endpoints of experiment 1 and 2. In vitro

maturation (IVM), in vitro fertilization (IVF), in vitro embryo

culture (IVC), hours post insemination (hpi) ……………………… 123

Figure 2 - Percentage of cleaved (A) and blastocyst/cleaved (B) after previous

exposure (only during in vitro maturation) to DXR, A. oncocalyx or

onco A, only. a,b,c Distinct letters represent significant differences

among treatments (P < 0.05) (experiment 1) ……………………… 126

Figure 3 - Percentage of cleaved (A) and blastocyst / cleaved (B) after

exposure (only during in vitro embryo culture) to DXR, A.

oncocalyx or onco A. a,b Distinct letters represent significant

differences among treatments (P < 0.05) (experiment 2) ………….. 128

LISTA DE TABELAS

PROPRIEDADES DA PLANTA AUXEMMA ONCOCALYX E SEU

PRINCÍPIO ATIVO ONCOCALYXONA A

Table 1 - Main results of the use of A. oncocalyx and Onco A in several

species and cell types ……………………………………………… 57


AFETAM A SOBREVIVÊNCIA IN VITRO E O DESENVOLVIMENTO DE

FOLÍCULOS PRÉ-ANTRAIS CAPRINOS INCLUSOS EM TECIDO

CORTICAL OVARIANO

Table 1 - Percentage (mean ± SEM) of morphologically normal caprine

preantral follicles before (non-cultured control) and after in vitro

culture for 1 or 7 days in α-MEM+ alone or α-MEM+ supplemented

with DMSO, BMP15, DXR, A. oncocalyx (1.2; 12 and 34 µg/ml)

or onco A (1, 10 and 30 µg/ml) ……………………………………. 79

Table 2 - Percentage (mean ± SEM) of primordial and growing caprine

preantral follicles before (non-cultured control) and after in vitro

culture for 1 or 7 days in α-MEM+ alone or α-MEM+ supplemented

with DMSO, BMP15, DXR, A. oncocalyx (1.2; 12 and 34 µg/ml)

or onco A (1, 10 and 30 µg/ml) ……………………………………. 80

EFEITO DA TOXICIDADE DA FRAÇÃO DA AUXEMMA ONCOCALYX E

DO PRINCÍPIO ATIVO ONCOCALYXONA A NO CULTIVO IN VITRO

DE FOLÍCULOS SECUNDÁRIOS E NA MATURAÇÃO IN VITRO DE

OÓCITOS CAPRINOS

Table 1 - Oligonucleotide primers used for PCR analysis of goat secondary

follicles ……………………………………………………………. 103

Table 2 - Percentage of morphologically intact secondary follicles, and

antrum formation after in vitro culture for 7 days in α-MEM+

(control) or supplemented with DMSO, DXR, A. oncocalyx or onco

A ………………………………………………..………………… 105

Table 3 - Follicular diameter (on day 0 and 7) and growth rate (mean ± SEM)

of isolated secondary follicles after in vitro culture in α-MEM+

(control) or supplemented with DMSO, DXR, A. oncocalyx or onco

A …………………………………………………………………... 105

Table 4 - PCNA test and TUNEL assay of non-cultured or in vitro cultured

isolated secondary follicles for 7 days in α-MEM+ (control) or

supplemented with DMSO, DXR, A. oncocalyx or onco A ………. 106

Table 5 - Viable and non-viable oocytes rates, germinal vesicle (GV),

meiotic resumption and metaphase II (MII) rates, after in vitro

maturation in TCM199+ (control) or supplemented with DMSO,

DXR, A. oncocalyx or onco A of COCs recovered from antral

follicles ……………………………………………………………. 107



DESENVOLVIMENTO OOCITÁRIO IN VITRO EM SUÍNOS, MAS SÃO

MENOS PREJUDICIAIS DO QUE A DOXORRUBICINA

Table 1 - Rates of viable oocytes, germinal vesicle (GV), meiotic resumption

and metaphase II (MII) rates, after in vitro maturation of porcine

oocytes in control medium alone or supplemented with DXR, A.

oncocalyx or onco A (experiment 1) …………………………..….. 124

Table 2 - Rates of viable oocytes, matured, penetrated, monospermy and

efficiency rates and number of spermatozoa per oocyte after

previous exposure (only during in vitro maturation) to DXR, A.

oncocalyx or onco A (experiment 1) ……………………………… 125

Table 3 Rates of viable oocytes, matured, penetrated, monospermy and

efficiency rates and number of spermatozoa per oocyte after 18hpi

exposure (only during in vitro embryo culture) to DXR, A.

oncocalyx or onco A (experiment 2) ………………………………. 127

LISTA DE ABREVIATURAS E SIGLAS

A. oncocalyx Auxemma oncocalyx

AgNOR

Argyrophilic proteins related to nucleolar organizer regions

(proteínas argirofílicas relacionadas com regiões organizadoras de

nucléolos)

ANOVA Analysis of variance (Análise de variância)

ANP Atrial natriuretic peptide

BAX BCL2 Associated X Protein

BCL2 B-cell lymphoma 2

BMP-15 Bone morphogenetic protein 15 (proteína morfogenética óssea 15)

BMPs Bone morphogenetic proteins

BSA Bovine serum albumin

CaCl2·2H2O Calcium chloride dehydrate

Calcein-AM Calcein acetoximetil

CAPES Coordenação de aperfeiçoamento de pessoal de nivel superior

Casp3 Caspase 3

cDNA Complementary DNA

cGMP Cyclic guanosine monophosphate

CH2Cl2 Dichloromethane

CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico

COCs / CCOs Cumulus oocyte complex (complexos cumulus-oócitos)

DAB 3,39-diaminobenzidine tetrahydrochloride

DMSO Dimethyl sulfoxide (dimetilsulfóxido)

DNA Deoxyribonucleic acid (ácido desoxirribonucléico)

DPBS Dulbecco’s phosphate-buffered saline

DSBs Double-strand breaks

dUTP Terminal deoxynucleotidyl transferase-mediated

DXR Doxorubicin (Doxorrubicina)

EGF Epidermal growth factor

EtOAc Ethyl acetate

EtOH Ethyl alcohol

FSH Follicle stimulating hormone

FUNCAP Fundação Cearense de Apoio ao Desenvolvimento Científico e

Tecnológico

GAPDH Glyceraldehyde-3-phosphate-dehydrogenase

GV Germinal vesicle

GVBD (RVG) Germinal vesicle break down (ruptura da vesícula germinativa)

H2O2 Hydrogen peroxide

HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

HL-60 Human promyelocytic leukemia line

hpi Hours post insemination

IGF-I Insulin growth factor I

IVF / FIV In vitro fertilization (fertilização in vitro)

IVM / MIV In vitro maturation (maturação in vitro)

IVP In vitro production

KCl Potassium chloride

LH Luteinizing hormone

mDPBS Modified DPBS

MEM Minimal essential medium (meio essencial mínimo)

MeOH Methanol

MI Metaphase 1

MII Metaphase 2

Na2HPO4 Sodium hydrogen phosphate

NaCl Sodium chloride

NCSU-23 North Carolina State University Medium-23

NMR Nuclear magnetic resonance

NOR Nucleolar organizer regions

Onco A Oncocalyxone A (oncocalyxona A)

PAF Paraformaldehyde

PAR Poly (ADP-ribose) chain

PARP Poly (ADP-ribose) polymerase

PCNA Proliferating cell nuclear antigen

PKG cGMP-dependent protein kinase

PPIA Peptidylprolyl Isomerase A

qPCR Quantitative PCR

rFSH Recombinant FSH

RNA Ribonucleic acid

RNAm Messenger RNA

S phase Synthesis

SEM Standard error of the mean

TCM-199+ Tissue culture medium 199

Topo II Topoisomerase II

TOPOs Topoisomerases

TPF TUNEL positive follicles

TUNEL Terminal deoxynucleotidil transferase-mediated deoxyuridine

Triphosphate biotin nick end-labeling

α-MEM Alpha minimal essential medium (meio essencial mínimo alfa)

SUMÁRIO

1 INTRODUÇÃO ......................................................................................... 20

2 REVISÃO DE LITERATURA ................................................................. 22

2.1 FOLICULOGÊNESE E CARATERIZAÇÃO FOLICULAR .................... 22

2.2 ATRESIA FOLICULAR ............................................................................. 24

2.3 TOXICIDADE E FUNÇÃO REPRODUTIVA FEMININA ...................... 24

2.4 SISTEMAS DE CULTIVO IN VITRO ........................................................ 26

2.4.1 Cultivo in situ e isolado de folículos pré-antrais ..................................... 26

2.4.2 Técnicas de avaliação do cultivo in vitro .................................................. 27

2.5 DOENÇAS TUMORAIS NEOPLÁSICAS ................................................ 29

2.5.1 Terapêutica do câncer ............................................................................... 29

2.6 FITOTERAPIA ........................................................................................... 30

3 JUSTIFICATIVA ...................................................................................... 33

4 HIPÓTESES CIENTÍFICA ...................................................................... 35

5 OBJETIVOS .............................................................................................. 36

5.1 OBJETIVO GERAL .................................................................................... 36

5.2 OBJETIVOS ESPECÍFICOS ...................................................................... 36

6

PROPRIEDADES DA PLANTA AUXEMMA ONCOCALYX E SEU

PRINCÍPIO ATIVO ONCOCALYXONA A .......................................... 37

7


AFETAM A SOBREVIVÊNCIA IN VITRO E O

DESENVOLVIMENTO DE FOLÍCULOS PRÉ-ANTRAIS

CAPRINOS INCLUSOS EM TECIDO CORTICAL OVARIANO ...... 59

8

EFEITO DA TOXICIDADE DA FRAÇÃO DA AUXEMMA

ONCOCALYX E DO PRINCÍPIO ATIVO ONCOCALYXONA A NO

CULTIVO IN VITRO DE FOLÍCULOS SECUNDÁRIOS E NA

MATURAÇÃO IN VITRO DE OÓCITOS CAPRINOS ......................... 84

9



DESENVOLVIMENTO OOCITÁRIO IN VITRO EM SUÍNOS, MAS

SÃO MENOS PREJUDICIAIS DO QUE A DOXORRUBICINA ........ 108

10 CONCLUSÕES .......................................................................................... 133

11 PERSPECTIVAS ....................................................................................... 134

REFERÊNCIAS ......................................................................................... 135

20

1 INTRODUÇÃO

As plantas são reconhecidas pelo seu potencial terapêutico e representam uma

excelente fonte de matéria-prima para a produção de novas drogas (Bahmani et al., 2016).

Assim, o estudo das atividades farmacológicas das plantas é de grande importância a fim

de provar sua eficiência e, em muitos casos, validar as suas utilizações populares (Schmitt

et al., 2003).

Dentre os diferentes tipos de plantas existentes, A Auxemma oncocalyx (A.

oncocalyx) tem sido amplamente utilizada na medicina popular no tratamento adjuvante

para ferimentos (Braga, 1976). Diversos estudos sugerem algumas atividades biológicas

desta planta, tais como atividade analgésica, antioxidante, anti-inflamatória e anti-tumoral

(Ferreira et al., 2004; Pessoa et al., 1992). O extrato da A. oncocalyx pode ser obtida a

oncocalyxona A (rel-8a-hydroxy-5-hydroxy-methyl-8ab-methyl-2-methoxy-7,8,8a,9-

tetrahydro-1,4 anthracenediona, onco A) que é o princípio ativo possuindo alta atividade

antioxidante (Ferreira et al., 2003) e antiproliferativa contra as células tumorais (Costa-

Lotufo et al., 2002a). A onco A tem sido sugerida como uma droga alternativa para o

tratamento do câncer (Barreto et al., 2013). No entanto, a maioria dos medicamentos

utilizados para a terapia de câncer tende a ser tóxico para a saúde reprodutiva feminina,

acarretando falha ovariana prematura (Turan et al., 2013). Entretanto, ainda não é

conhecido o efeito da Auxemma oncocalyx e do seu princípio ativo sobre a fertilidade

feminina, incluindo os seus possíveis efeitos sobre a sobrevivência e o desenvolvimento

dos folículos ovarianos. Uma forma de investigar este parâmetro é a utilização da técnica

de cultivo folicular in vitro.

O cultivo in vitro de folículos pré-antrais, também conhecido como "Ovário

artificial", é uma etapa importante na biotecnologia de manipulação in vitro de oócitos

inclusos em folículos pré-antrais (MOIFOPA). Esta biotecnologia é uma importante

ferramenta para a elucidação dos mecanismos básicos envolvidos na foliculogênese

ovariana (Arunakumari et al., 2010). Além disso, permite a realização de ensaios in vitro

para investigar os efeitos benéficos/tóxicos de drogas sobre os folículos ovarianos, antes

de sua utilização efetiva em experimentos com humanos e/ou animais. Vale salientar que

a utilização de material humano para experimentação laboratorial envolve várias questões

éticas. Neste sentido, alguns autores utilizam modelos animais para a pesquisa. Um

exemplo é o uso do tecido ovariano de cabra, a fim de verificar o efeito de diversas

21

substâncias sobre o desenvolvimento folicular, devido às semelhanças entre os ovários

dessa espécie com a humana (Faustino et al., 2011). Os suínos, são também modelos

animais muito utilizados para estudos de toxicidade sobre a maturação, fertilização e o

cultivo embrionário (Santos et al., 2014).

Para uma melhor compreensão da importância desta tese, na revisão de literatura

a seguir será realizada uma breve abordagem da foliculogênese e caracterização dos

folículos ovarianos, população e atresia folicular, cultivo in vitro de folículos pré-antrais

(ovário artificial), estado atual da técnica de MOIFOPA, diferentes aplicações do ovário

artificial, com ênfase na sua importância para o teste da eficiência/toxicidade de drogas

e, finalmente, a importância dos fitoterápicos utilizados no tratamento do câncer, com

ênfase na planta Auxemma oncocalyx e seu princípio ativo, onco A.

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2 REVISÃO DE LITERATURA

2.1 FOLICULOGÊNESE E CARACTERIZAÇÃO FOLICULAR

Na maioria das espécies, a foliculogênese é um evento iniciado ainda na vida pré-

natal e é definida como o processo de formação, crescimento e maturação folicular,

obedecendo à uma sequência de eventos característicos, que começam com o

estabelecimento da população folicular no ovário e termina com a ovulação. Durante esse

processo há uma intensa proliferação das células da granulosa e um aumento do volume

e diâmetro folicular, como resultado do acúmulo de água, íons, carboidratos e lipídios

(Amsterdam et al., 1989).

O folículo é considerado a unidade morfológica e funcional do ovário mamífero,

e tem duas funções principais: endócrina e gametogênica. A unidade folicular é composta

por um oócito circundado por células da granulosa e/ou tecais, sendo um elemento

essencial na promoção de um ambiente ideal para maturação, viabilidade, crescimento e

liberação de um oócito maduro no processo de ovulação (Cortvrindt and Smitz, 2001).

De acordo com o seu estágio de desenvolvimento, os folículos podem ser classificados

em pré-antrais (primordial, transição, primário e secundário) e antral (terciário e pré-

ovulatório) (Silva et al., 2004).

Os folículos primordiais são constituídos por um oócito primário (núcleo no

estágio de prófase da primeira divisão meiótica) circundados por uma camada de células

da pré-granulosa planas e uma membrana. Os folículos primordiais são os folículos de

menor tamanho e os mais numerosos no ovário mamífero (90% dos folículos) e

constituem a reserva de folículos quiescentes (Beckers et al., 1996). Eles podem ser

eliminados pelo processo de atresia ou continuar seu crescimento até ovulação

(McLaughlin and McIver, 2009).

Durante a fase inicial do crescimento dos folículos primordiais os folículos que

apresentam células da granulosa pavimentadas e cubicas são denominados de folículo de

transição (Silva et al., 2004). Aqueles que mantém seu crescimento ativo, se tornam

folículos primários, caracterizado por possuir um oócito circundado por uma camada de

células da granulosa cubóides (Picton, 2001).

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As células da granulosa cubicas continuam a dividir-se e formam camadas

concêntricas. Com duas ou mais camadas de células ao redor do oócito, os folículos são

denominados de folículos secundários. Próximo à parte externa da membrana basal,

proliferam as células da teca que darão origem à teca interna e externa que são

responsáveis, juntamente como oócito, pelo funcionamento do folículo (Young and

McNeilly, 2010).

Quando os folículos secundários crescem, inicia-se na sequência a formação de

uma cavidade antral dando origem aos folículos terciários, que após um período de

desenvolvimento, caso escapem da atresia, poderão dar origem aos folículos maduros pré-

ovulatórios ou folículos de Graaf (Picton, 2001). A formação do antro faz com que as

células da granulosa se diferenciem em dois grupos: as células da granulosa murais, que

estão em contato com a membrana basal e têm uma função endócrina, e as células do

cumulus, que estão intimamente relacionadas com o oócito, e formam o complexo-

cumulus-oócito (CCO), que auxiliam no metabolismo e na maturação oocitária

(Gonçalves et al., 2008).

Nos folículos antrais, o antro é repleto com fluido folicular que atua como fonte

de oxigênio, tampão ácido-básico, hidratos de carbono, fatores de crescimento,

hormônios e outras substâncias (Sutton et al., 2003). O oócito completa o seu crescimento

quando o folículo que o contém entra na fase antral avançada, mas continua a maturar

(maturação nuclear e citoplasmática) até o final da foliculogênese. No citoplasma, as

alterações que ocorrem são destinadas à aquisição da capacidade de ser fertilizado pelo

espermatozoide, bloquear a polispermia, descondensar a cromatina do espermatozoide e

permitir a formação dos pró-núcleos masculinos e femininos, bem como as primeiras

divisões embrionárias (Ferreira et al., 2009). A nível nuclear, uma série de eventos

moleculares associados ocorrem em cascata com a retomada da meiose I: ruptura da

vesícula germinativa (RVG), a progressão na divisão meiótica, extrusão do primeiro

corpo polar e metáfase II (MII) (Tripathi et al., 2010). Além disso, ocorre a expansão das

células do culmulus.

Finalmente, o processo de foliculogênese termina quando o CCO é liberado de um

folículo maduro por ocasião da ovulação. Após a ovulação, a parede folicular

remanescente o sofre um processo de luteinização formando corpo lúteo.

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2.2 ATRESIA FOLICULAR

O processo de atresia usualmente ocorre de forma diferenciada nos folículos pré-

antrais e antrais. Em folículos pré-antrais, os primeiros sinais de morte folicular surgem

no oócito, em que pode-se observar a retração da cromatina nuclear e a fragmentação

oocitária (Morita and Tilly, 1999). Após a formação do folículo antral ocorre uma

alteração na sensibilidade do oócito e das células da granulosa. A partir deste estágio, o

oócito torna-se mais resistente à atresia e as primeiras alterações indicativas de atresia são

observadas nas células da granulosa.

A atresia pode ocorrer por necrose e/ou apoptose (Saumande, 1981). A necrose é

iniciada por mecanismos não-celulares, tais como a isquemia, depleção de ATP (Bhatia,

2004) e fatores traumáticos ocasionados por alterações no fornecimento de oxigênio e

nutrientes para o ovário, que resultam em danos celulares irreversíveis (Mccully et al.,

2004).

Já a apoptose é um processo de morte celular ativo, que ocorre de forma ordenada

e demanda energia para a sua execução. Além disso, a ativação deste processo é

geneticamente determinada, ou seja, é regulada pela expressão de genes específicos

(Mccully et al., 2004), em que, provavelmente o desbalanço entre os fatores que

promovem a sobrevivência e aqueles que induzem a apoptose irá determinar quais os

folículos que continuarão o seu desenvolvimento ou sofrerão atresia (Bhatia, 2004). A

apoptose é mediada por mecanismos intrínsecos ou extrínsecos (Johnson, 2003), como

estresse oxidativo, irradiação, ativação de genes promotores da apoptose, danos no DNA,

citocinas, ou ausência de fatores de crescimento (Johnson, 2003). Outros fatores que

levam a apoptose folicular são os agentes quimioterápicos.

2.3 TOXICIDADE E FUNÇÃO REPRODUTIVA FEMININA

Empresas farmacêuticas e químicas produzem novos químicos na forma de novas

drogas para o tratamento de pacientes com câncer. Essas drogas podem interferir com a

síntese, secreção, transporte, ligação ou eliminação de hormônios naturais, e tem o

potencial para alterar o desenvolvimento reprodutivo e a fertilidade, levando a desordens

reprodutivas (Kort et al., 2014). Qualquer droga que atue como um agente tóxico

reprodutivo, tem um efeito direto no ovário e deve também ser capaz de alterar

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mecanismos epigenéticos no oócito, resultando em efeitos epigenéticos ao longo das

gerações (Kang and Roh, 2010).

O ovário mamífero contém folículos em diferentes estágios de desenvolvimento,

que variam sua susceptibilidade a diferentes compostos (Stefansdottir et al., 2014).

Durante a foliculogênese, os folículos ovarianos entram em um período de crescimento

contínuo até eles entrarem em atresia ou desenvolverem até o estágio de folículos

maduros, acompanhados por uma rápida proliferação das células da granulosa. O estado

de crescimento contínuo, acompanhado pela parada meiótica do oócito durante a vida

reprodutiva feminina, faz dos folículos alvos vulneráveis para tóxicos reprodutivos

(Stefansdottir et al., 2014). Embora a estrutura folicular atue como uma barreira protetora

envolvendo o oócito, ela não protege necessariamente dos efeitos mutagênicos diretos ou

indiretos (Rekhadevi et al., 2014). Alguns compostos tóxicos são capazes de passar

através da membrana basal e têm o potencial de afetar o oócito, direta ou indiretamente,

ao afetar as células somáticas. Se essas drogas são capazes de afetar o pool de folículos

primordiais, elas podem causar a falência ovariana pré-matura, levando à infertilidade

pela depleção da reserva ovariana de folículos primordiais (De Vos et al., 2010). Por outro

lado, se o alvo dessas drogas é folículos em crescimento, isso pode resultar em atresia

folicular com subsequentes distúrbios cíclicos, causando a depleção do pool de folículos

primordiais (Meirow et al., 2010).

Existem diferentes rotas pelas quais os químicos podem interferir no

desenvolvimento do oócito. Agentes quimioterápicos podem afetar o fuso do oócito

durante a divisão meiótica, causando alteração no cromossomo (Fragouli et al., 2011).

Também, eles podem causar erros nos checkpoints meióticos, resultando em mutações

que têm potencial para a formação de embriões com aneuploidia e provalmente aborto

(Barekati et al., 2008), ou em outros casos, passam para as gerações subsequentes

(Stefansdottir et al., 2014).

Compostos tóxicos podem tanto atuar especificamente no oócito, ou ter impactos

mais amplos sobre as células somáticas (Orisaka et al., 2009). Qualquer efeito sobre as

células da granulosa e da teca pode alterar a produção de hormônios, perturbando o eixo

hipotalâmico-hipofisário-gonadal e o desenvolvimento folicular normal, afetando a

maturação do oócito (Canipari, 2000).

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2.4 SISTEMAS DE CULTIVO IN VITRO

Diferentes sistemas de cultivo in vitro têm sido desenvolvidos com o objetivo de

promover o crescimento de folículos pré-antrais ou antrais até seu completo

desenvolvimento, originando oócitos fertilizáveis ( Jin et al., 2010; Eppig and O’Brien,

1996; Eppig and Schroeder, 1989). Esses cultivos têm se tornado de grande importância

para o estudo e compreensão da foliculogênese, bem como para avaliar o efeito de

diferentes substâncias. Essa biotecnologia tem sido estabelecida com sucesso em vários

modelos animais (camundongo, rato, vaca, ovelha, cabra, porca e primatas) ( Xuying et

al., 2011; McLaughlin and Telfer, 2010; Telfer et al., 2008; Hirao et al., 1994).

Modelos de cultivo in vitro de folículos pré-antrais na forma isolada ou inclusos

em fragmentos de tecido ovariano, ou mesmo no ovário inteiro, permitem controlar de

forma mais precisa do que os testes in vivo, o efeito de diferentes drogas sobre a

foliculogênese. Potenciais aplicações incluem o estudo dos mecanismos de ação das

substâncias tóxicas e como elas levam a danos no oócito e células somáticas, interferem

na qualidade do oócito, no estabelecimento do pool de folículos primordiais e nas

interações parácrinas (Sun et al., 2004). O cultivo também pode revelar o alvo de um

determinado composto sobre um estágio específico de desenvolvimento folicular,

permitindo avaliar se o referido composto afeta a integridade cromossômica do oócito,

ou se ele tem a capacidade de alterar a sinalização hormonal dentro e/ou entre os folículos

(Stefansdottir et al., 2014).

Os métodos de cultivo disponíveis para pesquisa toxicológica variam de acordo

com a espécie, estágio folicular, período e composição do meio de cultivo. Cada sistema

de cultivo tem suas vantagens e desvantagens, exigindo uma análise cuidadosa antes de

escolher o melhor método para um estudo de toxicologia (Stefansdottir et al., 2014).

2.4.1 Cultivo in situ e isolado de folículos pré-antrais

Os folículos podem ser cultivados “in situ”, ou seja, inseridos no córtex ovariano,

ou “isolados”. Em adição, o cultivo pode ser realizado em dois passos, podendo ser

iniciado com o cultivo de folículos in situ, seguido de uma etapa de cultivo de folículos

isolados (Brien et al., 2003; Telfer et al., 2008). O cultivo de pequenos fragmentos de

córtex ovariano tem sido realizado para o estudo da ativação e crescimento de folículos

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em diferentes espécies, como caprinos (Silva et al., 2004), bovinos (Braw-Tal and

Yossefi, 1997), babuínos (Wandji et al., 1997) e humanos (Zhang et al., 2004). Além da

praticidade, o cultivo in situ apresenta também como vantagem a manutenção do contato

celular (Abir et al., 2006) e da integridade tridimensional dos folículos. No entanto, neste

tipo de modelo, embora haja uma expressiva ativação folicular, poucos folículos

primários cultivados progridem até o estágio de folículo secundário (Fortune, 2003).

O cultivo de folículos isolados apresenta como vantagens a possibilidade do

acompanhamento individual dos folículos durante o cultivo, além de favorecer melhor a

perfusão do meio para o folículo (Abir et al., 2006).

Uma vez isolados, os folículos podem ser cultivados em sistema bidimensional ou

tridimensional. No sistema bidimensional, o folículo é colocado diretamente sobre uma

placa de cultivo (exemplo superfície plástica) (Gonçalves et al., 2008), ou sobre uma

camada de células somáticas ou de matriz extracelular (exemplo o alginato). Já na forma

tridimensional, os folículos são cultivados inseridos em uma matriz extracelular, como

por exemplo, o colágeno ( Hirao et al., 1994; Carroll et al., 1991) ou gel de alginato (Xu

et al., 2009; West et al., 2007), mantendo a morfologia folicular intacta.

O fato do folículo permanecer com a morfologia intacta é muito importante, uma

vez que existe uma comunicação entre o oócito e as células somáticas que o circundam

(Spears et al., 1994), através das junções do tipo gap, pelas quais circulam fatores

parácrinos essenciais para proporcionarem crescimento e maturação do oócito

(Carabatsos et al., 2000). A perda desse contato causa ovulação pré-matura e liberação de

oócito degenerado (Eppig et al., 2005).

2.4.2 Técnicas de avaliação da eficiência do cultivo in vitro

Há um grande interesse no desenvolvimento de tecnologias que permitam o

crescimento e maturação in vitro de oócitos inclusos em folículos pré-antrais (maior

população de folículos presentes no ovário), devido às muitas aplicações que podem ter

tanto na reprodução assistida em humanos, como na produção animal.

Para a avaliação da eficiência do cultivo in situ pode ser utilizada a histologia

clássica para a análise quantitativa de folículos inclusos em tecido ovariano, com a

finalidade de verificar modificações na morfologia das células da granulosa, de

28

pavimentosa para cúbica. Assim, a histologia clássica permite classificar os folículos pré-

antrais quanto ao seu estágio de desenvolvimento (primordial, transição, primário ou

secundário), e ainda, quanto às suas características morfológicas (normais ou atrésicos).

Outras técnicas, tais como imunohistoquímica, também são amplamente utilizadas na

análise do cultivo in situ. Estas técnicas possibilitam analisar a proliferação celular,

apoptose e a expressão de moléculas de interesse (Gonçalves et al., 2008). O PCNA e o

AgNOR são marcadores apropriados para estudar a proliferação das células da granulosa.

Uma técnica empregada para avaliar a viabilidade de folículos pré-antrais isolados

e oócitos após o cultivo in vitro é a microscopia de fluorescência (Silva et al., 2011; Bruno

et al., 2010), a qual utiliza marcadores fluorescentes, que quando excitados com radiação

de baixo comprimento de onda, absorvem energia e emitem luz de comprimento de onda

maior (Gonçalves et al., 2008). Os marcadores comumente utilizados são o etídio

homodímero-1 e calceína-AM, que permitem a detecção simultânea de células mortas e

vivas, respectivamente. O etídio homodímero-1 marca ácidos nucléicos em células não

viáveis, indicando a perda da integridade da membrana plasmática das células (Gonçalves

et al., 2008), enquanto a atividade enzimática (esterases) no citoplasma é detectada nas

células foliculares através do composto calceína-AM, que é clivado por enzimas esterases

em células vivas, resultando em um produto fluorescente (De Clerck et al., 1994). A

microscopia de fluorescência é empregada ainda no intuito de avaliar a configuração da

cromatina de oócitos oriundos de folículos pré-antrais, indicando assim o estágio meiótico

alcançado após terem sido cultivados e maturados in vitro (Saraiva et al., 2010)G. Para

tal finalidade, utiliza-se o Hoescht 33342, que penetra em células vivas e se intercala entre

as bases nitrogenadas do DNA.

A quantificação dos níveis de RNAm para as diferentes substâncias (ligantes e

receptores) que atuam durante a foliculogênese, também é considerada uma importante

ferramenta para auxiliar na compreensão desse processo, uma vez que permite detectar

alterações nos padrões de expressão gênica que ocorrem em resposta à fenômenos

relacionados à sobrevivência, ao crescimento e à diferenciação celular (Zamorano et al.,

1996). Dentre as técnicas comumente utilizadas para esta finalidade, pode-se destacar a

qPCR.

Outras ferramentas para a análise da eficiência do cultivo e da competência

oocitária são a fertilização in vitro (FIV) e a produção in vitro de embriões (PIV). A PIV

29

é uma excelente ferramenta para pesquisa de fenômenos biológicos que ocorrem durante

a maturação, fecundação e cultivo in vitro de oócitos, capacitação espermática e eventos

relacionados ao início do desenvolvimento embrionário na fase de pré-implantação.

Devido à sua capacidade de produzir um grande número de embriões, a PIV se tornou um

instrumento indispensável para outras biotécnicas como a clonagem, a manipulação de

genes e a transferência de núcleos (Gonçalves et al., 2007).

2.5 DOENÇAS TUMORAIS NEOPLÁSICAS

As doenças tumorais neoplásicas vêm sendo indicadas como a terceira causa de

morte mais frequente no Brasil (Machado and Melo-junior, 2009). No mundo, o câncer é

responsável por mais de 12 % de todos os óbitos, e é alvo de pesquisas para o

desenvolvimento ou descoberta de novas formas de tratamento (Saúde, 2008), uma vez

que a resistência adquirida aos medicamentos anticancerígenos já existentes é encontrada

em grande parte dos pacientes, além da ocorrência de inúmeros efeitos adversos que

limitam a eficácia do tratamento.

2.5.1 Terapêutica do câncer

Atualmente, o tratamento dos cânceres, em sua grande maioria, é considerado

como um dos problemas mais desafiadores da medicina. A partir do momento que a

neoplasia primária causa metástase pelo corpo do hospedeiro, o prognóstico se torna ruim,

sendo a quimioterapia antineoplásica a principal forma de tratamento neste estágio. Uma

vantagem deste tratamento é a de atingir igualmente as metástases disseminadas pelo

corpo. Entretanto, há desvantagens importantes a serem consideradas, principalmente

aquelas relacionadas aos seus efeitos colaterais, pois em sua grande maioria, estes

medicamentos apresentam baixo índice terapêutico, ou seja, dose terapêutica muito

próxima à dose tóxica (Chabner and Roberts, 2005). Desta maneira, a pesquisa, tanto

básica como aplicada, é fundamental e deve ser estimulada, para que medicamentos

antineoplásicos mais eficazes e seguros sejam descobertos, incluindo drogas que não

afetem a função reprodutiva, ou seja, não sejam tóxicas aos ovários.

Com o objetivo de tratar o câncer com maior eficácia, esquemas terapêuticos

utilizando cirurgia, radioterapia e quimioterapia têm sido cada vez mais prevalentes

30

(Reiner et al., 2009). Cada um destes tratamentos visa erradicar o câncer, normalmente

por meio da terapia combinada, em que é associado mais de um tipo de tratamento.

Através da cirurgia ou radioterapia, um terço dos pacientes consegue ser curado através

de medidas locais, que são eficazes quando o tumor ainda não sofreu metástase por

ocasião do tratamento. Todavia, nos demais casos, a neoplasia caracteriza-se pelo

desenvolvimento precoce de micrometástases, indicando a necessidade de uma

abordagem sistêmica, que pode ser efetuada, em cerca de 60-70% dos casos, com a

quimioterapia (Almeida et al., 2005). Esta medida terapêutica consiste na utilização de

medicamentos a fim de destruir as células cancerosas, bloqueando o seu

desenvolvimento. Entretanto, a maioria dos agentes quimioterápicos atua de forma não

específica, lesando tanto células malignas quanto normais (Almeida et al., 2005). Porém,

o corpo recupera-se destes inconvenientes após o tratamento, e o uso clínico desses

fármacos exige que os benefícios sejam confrontados com a toxicidade, na procura de um

índice terapêutico favorável (Almeida et al., 2005).

Muitas drogas usadas na quimioterapia do câncer, além de possuir toxicidade

contra células tumorais, exibem efeitos genotóxicos, carcinogênicos e teratogênicos sobre

células normais, revelando uma baixa especificidade dessas drogas contra os tecidos

tumorais, resultando em efeitos não desejáveis no tratamento. A Doxorrubicina (DXR) é

um fármaco amplamente utilizado em pacientes com câncer (Oktem and Oktay, 2007).

Estudos recentes revelam que a DXR induz toxicidade ovariana, que é observada pela

redução da taxa de ovulação, juntamente com uma redução no tamanho do ovário (Ben-

Aharon et al., 2010; Oktem and Oktay, 2007). Neste contexto, os compostos derivados

de plantas (fitoterápicos) também têm sido uma fonte alternativa de moléculas

clinicamente úteis no tratamento do câncer (Cragg and Newman, 2005).

2.6 FITOTERAPIA

A expressão fitoterapia é atribuída aos medicamentos originados exclusivamente

de material botânico integral ou seus extratos utilizados com o propósito de tratamento

médico (Dewick, 2009).

Antes a fitoterapia era utilizada principalmente por populações carentes, isso pelo

fato da boa disponibilidade e menor custo. A fitoterapia e predominante em países

31

emergentes, sendo bem estabelecida em culturas e tradições, especialmente na Ásia,

América Latina e África (Shale et al., 1999).

Existe um interesse crescente no uso de produtos naturais, principalmente os

derivados de plantas. De acordo com Gurib-Fakim (2006), os produtos naturais e seus

derivados representam mais de 50% de todas as drogas utilizadas no mundo, e as plantas

medicinais contribuem com 25% deste total. Alguns exemplos de drogas obtidas a partir

de plantas são a Digoxina da Digitalis spp., Quinina e Quinidina obtidas da Cinchona

spp., Vincristina e Vinblastina oriundas da Catharanthus roseus, Atropina procedente de

Atropa belladona e Morfina e Codeína provenientes de Papaver somniferum (Rates,

2001).

A possibilidade de utilizar as plantas medicinais como fonte de substâncias

medicamentosas reside na capacidade de produzirem, a partir de seu metabolismo,

substâncias químicas que exercem alguma atividade sobre outros organismos vivos.

Baseado nessas atividades e que se procuram os efeitos terapêuticos para o tratamento de

doenças tanto em animais como em humanos (Boukandou et al., 2015). As plantas podem

sintetizar dois tipos de metabólitos: primários e secundários. A extensa e diversificada

flora do Brasil e um recurso natural de imenso potencial para a obtenção de metabólitos

secundários, muitos dos quais podem ser utilizados com finalidade terapêutica (da Silva

et al., 2016). De acordo com Gurib-Fakim (2006), as atividades biológicas das plantas

são atribuídas a esses metabólitos. As propriedades antifúngica, antibacteriana,

antineoplasica, imunomoduladora e antiparasitaria das plantas têm sido bastante

exploradas (Silva et al., 2016).

Algumas dessas plantas têm uma longa história de uso no tratamento de câncer. O

maior impacto recente de drogas derivadas de plantas foi provavelmente nesta área, em

que Taxol, Vinblastina, Vincristina e Camptotecina melhoraram drasticamente a eficácia

da quimioterapia contra alguns dos piores cânceres. Na verdade, mais de 60% dos agentes

anticancerígenos utilizados são derivados da natureza (Cragg and Newman, 2005).

Apesar deste vasto conhecimento, ainda são limitadas as investigações cientificas visando

determinar o potencial terapêutico das plantas (Duarte, 2006).

Estudos têm demonstrado que as populações locais da região do semiárido

brasileiro possuem um vasto conhecimento sobre a utilidade medicinal de determinadas

espécies de plantas, como por exemplo, a Auxemma oncocalyx (A. oncocalyx), também

32

conhecida como Pau-Branco, o que pode contribuir para a conservação da biodiversidade

e sua função dentro do ecossistema (Albuquerque and Oliveira, 2007).

A Auxemma oncocalyx é uma planta nativa da caatinga do Nordeste brasileiro, e

vem sendo bastante utilizada pela medicina popular. Seu composto ativo oncocalyxona

A (onco A) é conhecida por ter diversas propriedades bioquímicas, como antioxidante,

anti-inflamatório, anti-plaquetário e anti-cancerígeno. Mais informações sobre a planta e

seu composto ativo encontrasse no artigo de revisão (Capítulo 1).

33

3 JUSTIFICATIVA

Os fitoterápicos sofrem uma grande restrição quanto ao seu uso e aceitação,

devido ao reduzido número de estudos que comprovam sua ação biológica e segurança

quanto a efeitos tóxicos agudos, crônicos ou sobre a reprodução (Sharapin, 1999). Dentre

os critérios de segurança necessários, estão os estudos sobre a toxicidade reprodutiva dos

produtos fitoterápicos, que incluem uma avaliação das ações sobre a fertilidade e a

performance reprodutiva para os produtos administrados, durante a gametogênese e

fecundação.

Vários métodos in vitro para avaliar a toxicidade de substâncias foram

padronizados utilizando-se cultivos celulares. Um dos modelos experimentais utilizados

para avaliação da toxicidade in vitro, que surge como alternativa previa aos estudos in

vivo, é a biotécnica de Manipulação de Oócitos Inclusos em Folículos Pré-Antrais

(MOIFOPA/ Ovário artificial). A MOIFOPA consiste no isolamento, conservação

(resfriamento e criopreservação) e/ ou cultivo in vitro de folículos pré-antrais, visando a

ativação, crescimento e maturação in vitro dos folículos primordiais até o estágio pré-

ovulatório. Além de ter importantes aplicações para a pesquisa fundamental e reprodução

assistida animal e humana, esta biotécnica também representa uma excelente alternativa

para incrementar e auxiliar no desenvolvimento de pesquisas relacionadas à indústria

farmacêutica (Figueiredo, 2008). Portanto, a importância do presente trabalho visa dar

continuidade às pesquisas relacionadas ao ovário artificial, testando a sua aplicação no

teste de toxicidade de substâncias de interesse terapêutico empregadas em humanos.

O uso de material humano é bastante problemático na pesquisa, devido a

dificuldades éticas, e até mesmo de obtenção de material de pesquisa. Assim, diversos

autores têm utilizado animais em suas pesquisas antes de sua aplicação em humanos. Um

exemplo disto é a utilização de tecido ovariano de cabras com objetivo de verificar o

efeito de diferentes substâncias no desenvolvimento folicular. Isso seria possível devido

ao fato desse animal ter uma foliculogênese semelhante à humana, além da própria

estrutura ovariana que também é semelhante (Amorim et al., 2004) Por outro lado, a

espécie suína representa um modelo ideal para o teste de toxicidade de competência

oocitária e qualidade embrionária. Em comparação com as outras espécies, a espécie

suína é um modelo animal bastante utilizado como um modelo para oócitos humanos em

34

testes de toxicidade (Gerritse et al., 2008; Munn et al., 1986). Além disso, a vantagem de

utilizar ovários de porcas é que os ovários são obtidos a partir de animais em idade

semelhante, raça e nutrição controlada.

Uma das principais vantagens da utilização de modelos animais in vitro e

possibilidade de testar substâncias como novos antibióticos, hormônios, fatores de

crescimento e quimio e fitoterápicos poderão ter seus efeitos testados (benéfico ou tóxico)

sobre os oócitos in vitro. A utilização desta biotécnica como método laboratorial para

testes de drogas traz importantes consequências para o bem-estar animal, uma vez que

milhares de animais serão poupados de serem utilizados em experimentos/ testes no que

concerne aos testes in vivo (Figueiredo, 2008).

Dentre as plantas medicinais estudas por diversos autores, podemos destacar a

Auxemma oncocalyx (Pau-Branco-do-Sertão). Conforme já descrito anteriormente, esta

planta possui diversas atividades biológicas. O extrato hidroalcoólico dessa planta

mostrou ação antitumoral, analgesica e antinflamatória (Ferreira et al., 2004; Lino et al.,

1996; Pessoa et al., 1992). Estudos de quinonas isoladas, como o caso da oncocalyxona

A, observaram fortes atividades como antiagregante plaquetaria, antioxidante,

antinflamatória, analgesica, antitumoral e antifungica (Sun et al., 2016; Lee et al., 2015;

Ferreira et al., 2003; Leyva et al., 2000). Além disso, essa planta vem sendo utilizada pela

medicina popular por possuir diversas propriedades medicinais. A casca, por exemplo, e

muito utilizada para auxiliar na cicatrização de ferimentos (Pessoa, 1994; Braga, 1976).

Entretanto, apesar desses vários estudos, ainda não é conhecido o efeito dessa planta sobre

a fertilidade feminina.

35

4 HIPÓTESE CIENTÍFICA

Diante do exposto, foi formulada a seguinte hipótese científica:

Auxemma oncocalyx e onco A apresentam um efeito menos tóxico do que o

controle (tóxico) positivo DXR sobre a sobrevivência e crescimento folicular (espécie

caprina), maturação oocitária (espécies caprina e suína) e desenvolvimento

embrionário inicial in vitro (espécie suína).

36

5 OBJETIVOS

5.1 OBJETIVO GERAL

Avaliar o efeito da Auxemma oncocalyx e seu composto ativo a oncocalyxona A

sobre o cultivo in vitro de folículos pré-antrais, maturação oocitária e qualidade

embrionária.

5.2 OBJETIVOS ESPECÍFICOS

Avaliar o efeito da concentração-resposta da A. oncocalyx (1,2; 12 ou 34 μg/ml) e

onco A (1; 10 ou 30 μg/ml) sobre a sobrevivência, ativação, crescimento folicular e

oocitário após o cultivo in vitro de folículos pré-antrais caprinos inclusos em fragmentos

de tecido ovariano (Fase I);

Investigar o efeito da A. oncocalyx e onco A, utilizando as concentrações definidas

na Fase I, sobre a sobrevivência folicular, formação de antro e o crescimento de folículos

secundários isolados caprinos cultivados in vitro, bem como sobre a viabilidade e a

maturação nuclear de oócitos recuperados de folículos antrais caprinos (Fase II);

Avaliar o efeito da exposição da A. oncocalyx e onco A durante a maturação in

vitro de oócitos (Experimento 1) ou durante o cultivo in vitro de embriões (Experimento

2) sobre a maturação oocitária desenvolvimento embrionário inicial na espécie suína

(Fase III).

37

6 PROPRIEDADES DA PLANTA AUXEMMA ONCOCALYX E SEU PRINCÍPIO

ATIVO ONCOCALYXONA A

“Properties of the plant Auxemma oncocalyx and its active principle oncocalyxone A”

Periódico: Phytotherapy research (submetido) (ISSN: 1099-1573)

Factor de impacto: 2.694

38

RESUMO

Atualmente, existe uma constante necessidade de pesquisar novas drogas, sendo as

plantas cada vez mais reconhecidas por seu potencial terapeutico. Assim, o estudo das

atividades farmacológicas de plantas é crucial para identificar sua eficácia, e em muitos

casos, validar a utilização popular de plantas medicinais. Auxemma oncocalyx (A.

oncocalyx) é uma árvore pertencente à família Boraginaceae, endêmica do nordeste do

Brasil. Esta planta vem sendo amplamente utilizada na medicina popular, e seus extratos

hidroalcóolicos contém principalmente a Oncocalixona A (Onco A). Nesta revisão, serão

abordadas a ecologia e biologia de A. oncocalyx. Adicionalmente, as propriedades

biológicas de A. oncocalyx e Onco A, como ação anti-inflamatória e analgésica, efeito

antiplaquetário e antioxidante, toxicidade e potencial anticancerígeno também serão

discutidas.

Palavras-chave: Auxemma oncocalyx. Oncocalyxona A. toxicidade. Terapia de câncer.

Propriedades biológicas.

39

Title: Properties of the plant Auxemma oncocalyx and its active principle Oncocalyxone

A

Leiva-Revilla J. *1, Lunardi F. O. 1, Araújo V. R. 1, Celestino J. J. H. 2, Rodrigues A. P.

R. 1, Figueiredo J.R1.

1 Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, Universidade Estadual do

Ceará, Fortaleza-CE, Brazil. Av. Paranjana, 1700. Itaperi. 60740000 - Fortaleza, CE –

Brasil. Telephone: (+55 85) 31019852

2 Institute of Health Sciences, Universidade da Integração Internacional da Lusofonia

Afro-Brasileira. Acarape-CE, Brazil. Rodovia CE 060 Km51. 62785000 - Acarape, CE –


* Corresponding author; Av. Paranjana, 1700. Itaperi. 60740000 - Fortaleza, CE – Brasil.

Telephone: (+55 85) 31019852. Fax: (+55 85) 31019840. e-mail address:

[email protected]

mailto:[email protected]

40

ABSTRACT

Nowadays, there is a constant need to search for new drugs, and plants have become

increasingly recognized for their therapeutic potential. Thus, the study of the

pharmacological activities of plants is crucial to verify the effectiveness and, in many

cases, to validate the popular uses of these medicinal plants. Auxemma oncocalyx (A.

oncocalyx), is a tree that belongs to the Boraginaceae family, and it is native of the

northeast of Brazil. This plant has been widely used in folk medicine and the

hydroalcoholic extract contains its principal component, the Oncocalyxone A (Onco A).

In this review, the ecology and biology of the A. oncocalyx is evaluated. In addition, the

biological properties of A. oncocalyx and Onco A, such as their anti-inflammatory and

analgesic action, antiplatelet and antioxidant effect, toxicity and anticancer potential are

also discussed.

Keywords: Auxemma oncocalyx, Oncocalyxone A, toxicity, cancer therapy, biological

proprieties

41

1. Introduction

Plants have become increasingly recognized for their therapeutic potential,

representing an excellent source of raw material in the production of new drugs (Gossell-

Williams et al., 2006). Thus, the study of the pharmacological activities of plants is of

paramount importance in order to prove the effectiveness, and in many cases to validate

the popular uses of these medicinal plants (Schmitt et al., 2003).

Among these plants we can quote Auxemma oncocalyx, popularly known as "Pau-

Branco", which is a tree that belongs to the Boraginaceae family. It is a plant native of

the Caatinga (semi-arid forest occurring only in Brazil) (Maia, 2004), and it is mainly

found in the state of Ceará and Rio Grande do Norte (Braga, 1976). This plant has been

widely used in folk medicine. The shell, for example, is widely used in the adjunctive

treatment of injuries such as wound healing (Braga, 1976; Pessoa, 1994). Moreover, it

has a forage value as food for vertebrates (caprine, murine and birds) and invertebrates

(Coleoptera, Diptera, Lepidoptera and Hemiptera), mainly due to the high protein and

lipid content of its fruits (Tigre, 1970). Additionally, this plant has a high ornamental

value, particularly in afforestation, and agroforestry, being used as a windbreak crop. It

is also used in reforestation of degraded areas (Maia, 2004), besides being used in

woodworking.

The hydroalcoholic extract of the stem has shown an antitumoral, analgesic,

antioxidant and anti-inflammatory action (Ferreira et al., 2004, 2003; Lino et al., 1996).

On the other hand, the branches have presented properties against Trypanosoma spp.

(Braga, 1976).

Pharmacological studies have shown an antiplatelet action and vasoconstriction

in conductance vessels from the methanolic extract of the heartwood of the stem. This

42

action is attributed to its active principle, the Oncocalyxone A (Onco A) (Sousa et al.,

2002).

Pessoa and Lemos (Pessoa and Lemos, 1997) isolated allantoin from the

hydroalcoholic extract of the A. oncocalyx. Furthermore, the rel-hydroxy-8a-hydroxy-5-

methyl-8ab-methyl-7-methoxy-2,8.8-0.9-tetrahydro-1,4-anthracenedione, also known as

Onco A, which represents 80% of the A. oncocalyx fraction (Pessoa et al., 1995, 1993),

was also isolated from the same extract. Other components and derivatives such as 6-

chloro-oncocalixona A, 11-O-acetyl-oncocalyxone A, and 8,11-O-diacetyl-oncocalyxone

have been isolated from the ethanol extract (Pessoa et al., 2004). Several studies have

shown that Onco A and these derivatives have a cytotoxicity against tumor cells (Pessoa

et al., 2004, 2003).

After the increasing rate of studies about the A. oncocalyx and its isolated

compounds, it becomes necessary to know more about the biological properties of this

plant. Given the importance of this plant as a possible chemotherapeutic drug, in the

present work we review the most relevant outcomes obtained to date, with an overview

of the biology of the plant and its biological properties.

2. Ecology and biology of Auxemma oncocalyx

A. oncocalyx (Boraginaceae) is a common tree found in the state of Ceará and Rio

Grande do Norte, Northeast of Brazil (Braga, 1976). This tree has between 6 to 8 meters

tall whereas the diameter of the trunk is 30 to 40 cm. It is characterized by small, dense

and white flowers, being this a peculiarity that attributes to its popular name (Maia, 2004).

The leaves are simple, alternate, elliptic, serrated from the middle to the apex and with

membranous consistency. Its fruits are glabrous drupes of 2.5 cm (Lima, 1989). Nozella

43

(Nozella, 2006) studied the nutritional value of native tree-shrub species of the semi-arid

region of northeast of Brazil, which are considered an important source of proteins for

the animals of this region, mainly during the dry season. Among these species, he

evaluated the A. oncocalyx, and according to the results of tannin concentrations, it has

been considered safe for animal nutrition (Nozella, 2006). Previous investigations of

Auxemma species resulted in isolation of eight compounds classified as cordiachromes

(Pessoa et al., 1995) and hydroquinones (da Costa et al., 1999; Pessoa et al., 1995, 1993).

The cordiachromes are a class of meroterpenoids present in A. oncocalyx and other plants

such as Auxemma glazioviana (da Costa et al., 1999). Some authors suggest that this

cordiachromes have antimycobacterial activity (Dettrakul et al., 2009), which helps the

tree against fungi and termite infestation (da Costa et al., 1999). On the other hand,

hydroquinones play an important role in electron transport, photosynthesis, and also, they

act as antioxidants (Dewick, 2009). Marques et al. (Marques et al., 2000) reported the

isolation and structural elucidation based on spectral analysis of three anthracene

derivatives such as auxenone, oncocalyxonol and auxemim.

The quinone fraction (QF) is prepared from the ground heartwood methanolic

extract through exhaustive aqueous extraction followed by lyophilization. The

hydrosoluble fraction contained around 80% of Onco A (Ferreira et al., 2004; Pessoa et

al., 1993), which was previously characterized in another study (Pessoa et al., 1993).

Allantoin is isolated from the hydroalcoholic extract of the stem (Pessoa et al., 1995)

Allantoin is a compound that has various pharmacological activities, like wound healing,

and stimulating cellular mitosis and promoter of epithelial stimulation. Allantoin is very

used in cosmetic and pharmaceutical preparations (Qunaibi et al., 2009). Araújo et al.,

(Araújo et al., 2010) suggested that the mechanism induced by allantoin in the wound

44

healing process occurs via the regulation of inflammatory response and stimulus of

fibroblastic proliferation and extracellular matrix synthesis.

Many biological effects have been attributed to this plant, and most of them relates

to the Onco A. So far, studies have presented various properties of Onco A, such as anti-

inflammatory, antiplatelet, antioxidant and anti-carcinogenic as described in Table 1.

These properties will be better described below.

3. Biological proprieties of Auxemma oncocalyx and Oncocalyxone A

3.1 Anti-inflammatory and analgesic

Although widely used by the population as an anti-inflammatory and analgesic

drug, few scientific reports exist testing the action of A. oncocalyx for these purposes.

The quinones present in the fraction of A. oncocalyx, which contains 80% of Onco A

(Pessoa et al., 1993), are possibly responsible for the analgesic and anti-inflammatory

activity.

The reduction of induced paw edema and inhibition of induced abdominal

contractions in mice was observed in a dose dependent-manner (1 and 5 mg/kg body) of

QF, indicating an action on the inflammatory process, possibly due to the blockade of

prostaglandin synthesis (Ferreira et al., 2004). In the same experiment, the analgesic

ability of the QF was evaluated by an induction of acetic acid in mice and through the use

of the formalin test, which is considered as a good model for chronic pain (Dubuisson

and Dennis, 1977). It was observed that the acetic acid produced a painful reaction with

an acute inflammation in the peritoneal area. This study showed that, when these animals

were treated with the QF of A. oncocalyx, there was a dose-dependent inhibition of

45

abdominal contractions induced by acetic acid, and a positive response to formalin, thus,

showing the analgesic properties of the QF of A. oncocalyx.

3.2 Antiplatelet and antioxidant

The antiplatelet effect of Onco A has been previously described (Ferreira et al.,

2008, 1999). Ferreira et al. (Ferreira et al., 2008) tested the effect of Onco A in human

platelet cells, and showed that there was a reversal inhibitory effect concentration-

dependent. According to these authors, Onco A inhibits ATP liberation, and increase

cGMP levels without affecting cAMP and nitric oxide (NO) levels, causing the inhibition

of platelet aggregation. Onco A inhibited induced-aggregation probably by interfering

with a similar step of platelet activation (Haslam et al., 1999). However, Onco A had no

effect in NO production in human platelets, suggesting that the increase in cGMP levels

observed in this study was not dependent on a NO mechanism. This information suggests

that Onco A might by acting in two ways, downstream of the activation of soluble

guanylate cyclase (sGC) or, in a similar way of NO-independent activators of sGC, which

act by a synergistic mechanism that combine an increased production and reduced

degradation of cGMP (Ferreira et al., 2008).

Another reported action of Onco A, although with fewer studies, is its antioxidant

activity. Recently, interest in antioxidants action in biological systems has grown steadily,

possibly because it is associated with preventing tissue damage and release of free radicals

(Saeidnia and Abdollahi, 2013). In general, antioxidants are substances that prevent

deleterious damage from oxidation by inhibiting lipid peroxidation, or by sequestering

free radicals and chelating metal ions (Ferreira et al., 2003).

Toxicological studies have shown that synthetic antioxidants are capable of

causing harmful effects in animals and humans (Bouayed and Bohn, 2010). Thus, this

46

indicates the importance of studying the use of natural antioxidants, such as Onco A, in

biological systems.

The QF hepatotoxicity in rats was evaluated by induction with Carbon

tetrachloride (CCl4). CCl4 is known to induce intoxication through the production of free

radicals and lipid peroxidation. Mice treated with the QF showed a higher

hepatoprotective activity, possibly because of its antioxidant activity (Ferreira et al.,

2003). Onco A caused an inhibition of the cytochrome P-450 activity. Moreover, it was

able to stop the lipid peroxidation process, stabilized the hepatocellular membrane, and

improve protein synthesis (Ferreira et al., 2003). From all these data, Ferreira et al.

(Ferreira et al., 2008) suggested that Onco A could be a promising component for the

development of anti-thrombotic drugs.

3.3 Toxicity

Several authors reported the toxic effect of A. oncocalyx and Onco A. The effect

of different concentrations (1 – 100 µg/mL) of the quinone fraction of A. oncocalyx

(containing 80% of Onco A) on sea urchin eggs demonstrated that QF was capable of

inhibiting the cleavage of these eggs in a concentration-dependent manner. The beginning

of the destruction of the embryos started at the blastocyst stage with a concentration of

10 µg/mL, and there was a total destruction of the embryos (100%) with a concentration

of 30 µg/mL (Costa-Lotufo et al., 2002b). In another study, the cytotoxicity of Onco A

and other compounds in CEM leukemia cells and SW1573 lung tumor cells using

concentrations between 1 and 18 µg/mL was verified, demonstrating that all the

compounds caused cell death and inhibition of cell growth at concentration above 5

µg/mL in CEM cells. DNA damage of CEM cells was seen at concentrations of 5 µg/mL

for all four compounds, and a significant deleterious effect on DNA was observed as from

47

a concentration of 2 mg/mL of Onco A (Pessoa et al., 2004). Another study evaluated the

effect of different substances, among them Onco A and Onco C, in different

concentrations (0.4; 0.8; 1.6; 3.1; 6.2; 12.5 e 25 µg/mL) on human cells lines (CEM

leukemia, SW1573 lung tumor and CCD922 normal skin fibroblasts). This study showed

that both of them produced cytotoxicity with mean half maximal inhibitory concentration

(IC50) values of 0.8–2, 7–8 and 12–13 mg/mL against CEM, SW1573 and CCD922

respectively (Pessoa et al., 2000). Leyva et al. (Leyva et al., 2000) showed that

oncocalyxones A and C, both isolated from A. oncocalyx, were also cytotoxic to

multidrug resistant lung tumor cell lines (SW 1573, SW1573-S1, SW 1573-2R160),

which were moderately or even highly resistant to the conventional anticancer drugs

doxorubicin (DXR) and mitoxantrone. The cytotoxicity associated with these compounds

can be attributed to redox cycling and subsequent development of oxidative stress (Monks

and Lau, 1992). Thus, the cellular damage can occur by DNA alkylation (Bolton et al.,

2000). Consequently, quinones have a high toxicity related to their antimitotic properties.

Pessoa et al. (Pessoa et al., 2003) showed that the Onco A at the concentration of 0.5

g/mL was similar to 0.01 µg/mL DXR with regard to cytotoxicity in lymphocytes.

Lima (Lima, 2008) studied the effect of an aqueous extract of the leaves of A.

oncocalyx on the growth of bacterial culture. The study showed that this extract inhibited

the growth of gram-positive microorganisms, such as Bacillus subtilis and

Staphylococcus aureus. This antibacterial effect showed another toxic capacity of the A.

oncocalyx (Lima, 2008).

3.4 Anticancer

Several studies have reported the antitumoral activity of A. oncocalyx fraction and

Onco A. Moraes et al. (Moraes et al., 1997) was the first one to report the antitumoral

48

activity of the hidroalcoholic extract of A. oncocalyx. The study evaluated the anti-

cancerigenous effect of the extracts of 72 samples of plant species from northeastern

Brazil. Among many of the studied plants, A. oncocalyx showed an inhibitory activity

against Walker malignant tumor cells.

Other studies compared the cytotoxicity of Onco A with two conventional

anticancer agents, DXR and mitoxantrone, in a panel of human tumor cell lines. They

found that the IC50 was between 1.2 + 0.5 and 18 + 3.2 µg/mL depending on the cultured

cell line, and when multidrug resistant cell type were tested, Onco A showed a very high

potency when compared with DXR and Mitoxantrone (Leyva et al., 2000).

Pessoa et al. (Pessoa et al., 2000) showed that Onco A had an antitumoral activity

at a concentration between 0.8-2 µg/mL in leukemia cells. The antitumoral activity was

verified as the ability of Onco A to inhibit tumor cell line proliferation in the MTT [3-

(4’-5’- dimethylthiazol-2’-yl)-2.5-diphenyl-tetrazolium bromide] assay and this

cytotoxicity was related to the induction of DNA damage and the inhibition of DNA

synthesis. Finally, these authors suggested that this compound had moderate anticancer

potential.

There are several methods for testing genotoxic and cytotoxic potential. The

cytogenetic analysis represents an ideal tool to determine the DNA damage induced by

interaction of cells with exogenous substances. DNA damage can induce chromosomal

aberrations that can be used as mutagenic markers (Bahia et al., 1999).

Most anticancer compounds act at lower concentrations i.e., 1 µg/mL or 1 µM,

depending on the cultured cell line (Pessoa et al., 2000). Onco A can act between 0.8 and

18 µg/mL (Leyva et al., 2000; Pessoa et al., 2004, 2000). However, in cytotoxicity level,

derivatives from A. oncocalyx fraction can be less cytotoxic and less reactive against

49

leukemia cell DNA (Pessoa et al., 2004) than DRX, which is a widely used drug in cancer

treatment (Minotti et al., 2004).

To try to explain the effect of Onco A on cell growth, Pessoa et al. 15 evaluated

this effect in different phases of lymphocyte cell division (G1, G1/S and S). Onco A

showed similar cytotoxic results in G, G1/S and S phases when compared with DXR, but

only DXR showed genotoxicity. Genotoxicity can cause the occurrence of DNA

alterations leading to the possible formation of other tumors. The effect of Onco A was

seen in the DNA synthesis, in the transition G1/S. These results suggest that Onco A

possible interfere in the mechanisms involved in cell division and in the formation of the

spindle (Pessoa et al., 2003).

Many drugs used in cancer chemotherapy, in addition to having toxicity against

tumor cells, exhibit genotoxic, carcinogenic and teratogenic effects on normal cells,

showing a low specificity of these drugs against tumor tissue resulting in undesirable

effects of the treatments. For example, recent studies reveal that DXR induced ovarian

toxicity, which is observed by the reducing of the ovulation rate, together with a reduction

in the size of the ovary (Bar-Joseph et al., 2010; Ben-Aharon et al., 2010; Oktem and

Oktay, 2007). It its known that DXR elicits apoptosis by various mechanisms in a variety

of cells. It can be accumulated in both nucleus and mitochondria and induce chromosomal

obliteration by inhibiting topoisomerase-II. DXR can also interfere with mitochondrial

function and initiate an intrinsic pathway of apoptosis via the mitochondria by reducing

the mitochondrial membrane potential (MMP) and releasing cytochrome C (Bar-Joseph

et al., 2010) on cytoplasmic space. A oncocalyx and Onco A can be acting in a similar

pathway of DXR, but without causing genotoxicity (Pessoa et al., 2003), which may lead

to a more effective anticancer drug.

50

Barreto et al. (Barreto et al., 2013) described a new magnetic nano-system for

cancer therapy (MO-20), which facilitates the release of drugs at specific sites. This

system uses Onco A as a possible anticancer drug. The results showed potential future

applications of this technology (MO-20) with Onco A for cancer treatments.

4. Final Considerations

There are several properties of A. oncocalyx and Onco A, like anti-inflammatory,

analgesic, antiplatelet, antioxidant, cytotoxic and antitumor. It is important to emphasize

that Onco A has a cytotoxic effect on cancer cells in vitro without causing genotoxicity.

However, a study showed that Onco A caused embryo destruction and inhibition of

proliferation of tumor cells. Thus, further studies are needed to better understand the

mechanisms of A. oncocalyx and Onco A, especially in the field of anticancer activity and

toxic effect on organs and cells.

5. Conflicts of Interest

We wish to confirm that there are no known conflicts of interest associated with

this publication and there has been no significant financial support for this work that could

have influenced its outcome.

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57

Table 1. Main results of the use of A. oncocalyx and Onco A in several species and cell

types

Substance Researched

form

Tested

species Cell type Main result Reference

A. oncocalyx In vivo Rat Animal

analgesic and

anti-

inflammatory

activity

Lino et al.,

1996

A. oncocalyx In vitro Human

Walker

malignant

tumor cells

Anti-tumor

activity

Moraes et al.,

1997

A. oncocalyx In vitro Rat Hepatocytes

Decrease in

serum GOT and

GPT levels

(hepatoprotective

effect) and

Inhibition of

platelet activation

Ferreira et

al., 1999

Onco A In vitro Human Tumor cells Cytotoxic activity Leyva et al.,

2000

Onco A In vitro Human

CEM

leukemia,

SW1573 lung

tumour and

CCD922

normal skin

fibroblasts.

Antiproliferative

activity

Pessoa et al.,

2000

Onco A In vivo Rat Brain Inhibiting

lipoperoxidation

Ferreira et

al., 2001

A. oncocalyx In vivo Sea urchin Eggs

Concentration-

dependent

inhibition of sea

urchin eggs

development

Costa-Lotufo

et al., 2002

Onco A In vitro Human Platelet cells

Inhibits human

platelet

aggregation

Sousa et al.,

2002

Onco A In vitro Human lymphocytes Cytotoxic but not

genotoxic activity

Pessoa et al.,

2003

A. oncocalyx In vivo Mice and rats Animal Antioxidant

activity

Ferreira et

al., 2003

A. oncocalyx In vivo Mice Animal

Anti-

inflammatory and

antinociceptive

activity

Ferreira et

al., 2004

Onco A

In vitro

Human

Leukaemia

cells

Cytotoxicity

Pessoa et al.,

2004

58

A. oncocalyx In vitro Bacteria

Bacillus

subtilis,

Enterobacter

aerogenes,

Escherichia

coli, Klebsiella

pneumoniae,

Pseudomonas

aeruginosa,

Salmonella

cholerasuis and

Staphylococcus

aureus

Inhibition of

gram-positive

bacteria

Lima., 2008

Onco A In vitro Human Platelet cells

Inhibits human

platelet

aggregation by

increasing cGMP

and by binding to

GP Ibα

glycoprotein

Ferreira et

al., 2008

59

7 FRAÇÃO DE AUXEMMA ONCOCALYX E ONCOCALYXONA A AFETAM A

SOBREVIVÊNCIA IN VITRO E O DESENVOLVIMENTO DE FOLÍCULOS

PRÉ-ANTRAIS CAPRINOS INCLUSOS EM TECIDO CORTICAL

OVARIANO

“Fraction of Auxemma oncocalyx and Oncocalyxone A affects the in vitro survival and

development of caprine preantral follicles enclosed in ovarian cortical tissue”

Periódico: Forschende Komplementärmedizin (aceito) (ISSN: 1661-4119)

Qualis B2

60

RESUMO

Introdução: A. oncocalyx e seu componente principal (Onco A) possuem fortes ações

antioxidantes e antitumorais, entretanto, não existem estudos a respeito da ação de ambas

as substâncias durante a foliculogênese. Materiais e Métodos: Fragmentos de tecido

ovariano caprino foram fixados (controle fresco não cutivado) ou cultivados por 1 ou 7

dias em α-MEM+ isolado (controle cultivado) ou supementado com DMSO (0.5% v/v),

BMP-15 (100 ng/ml), doxorrubicina (DXR; 0.3 ug/ml) ou diferentes concentrações de A.

oncocalyx (1.2, 12 ou 34 µg/ml) ou Onco A (1, 10 ou 30 µg/ml). Foi avaliada a morfologia

e crescimento folicular, apoptose (ensaio de TUNEL), proliferação celular (AgNOR e

PCNA). Resultados: A. oncocalyx e Onco A (de modo concentração-dependente) e DXR

reduziram (P<0,05) a quantidade de folículos morfologicamente normais, sem efeito

(P>005) sob o crescimento folicular. A. oncocalyx reduziu o percentual de folículos

normais quando comparado ao grupo onco A (P<0,05). DXR, A. oncocalyx 1,2 e Onco A

1 aumentaram (P<0,05) o percentual de folículos marcados positivamente para TUNEL.

DXR reduziu significativamente (P>0,05) o número de regiões organizadoras de

nucléolos. Conclusão: A. oncocalyx e onco A afetaram o desenvolvimento folicular in

vitro no modelo caprino de modo dose-dependente. Onco A (1 µg/ml) possuem menor

efeito nocivo quando comparado a DXR na sobrevivência de folículos pré-antrais

caprinos.

Palavras-chave: Auxemma oncocalyx. Boraginaceae. Cultivo in vitro. Oncocalyxona A;

Folliculogenesis. Tecido cortical.

61

Title: Fraction of Auxemma oncocalyx and Oncocalyxone A affects the in vitro survival

and development of caprine preantral follicles enclosed in ovarian cortical tissue

Short title: “Effect of Auxemma oncocalyx and Oncocalyxone A on preantral follicles”

Leiva-Revilla J.1,*, Lima L.F. 1, Castro S.V. 1, Campello C.C. 1, Araújo V.R. 1, Celestino

J.J.H. 2, Pessoa O.D.L. 3, Silveira E.R. 3, Rodrigues A.P.R. 1, Figueiredo J.R. 1

1 Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, Universidade Estadual do

Ceará, Fortaleza-CE, Brazil. Av. Paranjana, 1700. Itaperi. 60740000 - Fortaleza, CE –


2 Institute of Health Sciences, Universidade da Integração Internacional da Lusofonia

Afro-Brasileira. Acarape-CE, Brazil. Rodovia CE 060 Km51. 62785000 - Acarape, CE –


3 Departamento de Química Orgânica e Inorgânica. Universidade Federal do Ceará,

Centro de Ciências, Av. Mister Hull S/N Pici 60455-760 - Fortaleza, CE - Brasil - Caixa-

postal: 12200 Telephone: (+55 85) 33669441

* Corresponding author; Av. Paranjana, 1700. Itaperi. 60740000 - Fortaleza, CE – Brasil.

Telephone: (+55 85) 31019852. Fax: (+55 85) 31019840. e-mail address:

[email protected]

mailto:[email protected]

62

ABSTRACT

Background: Auxemma oncocalyx (A. oncocalyx) and its main component

Oncocalyxone A (onco A), have a high level of antioxidant and anti-tumoral activity, but

there are no studies on the action of both of these drugs regarding folliculogenesis.

Materials and methods: Caprine ovarian tissue fragments were fixed (non-cultured

control) or cultured for 1 or 7 days in α-MEM+ alone (cultured control) or supplemented

with DMSO (20% v/v), BMP-15 (100 ng/ml), doxorrubicin (DXR; 0.3 g/ml) or different

concentrations of A. oncocalyx (1.2, 12 or 34 µg/ml) or onco A (1, 10 or 30 µg/ml). We

analyzed follicular morphology and growth, apoptosis (TUNEL assay), cell proliferation

(AgNOR and PCNA) Results: A. oncocalyx and onco A (concentration-dependent

manner) and DXR decreased (P<0.05) morphologically normal follicles, with no effect

(P>005) over follicular growth. A. oncocalyx reduced (P<0.05) the percentage of normal

follicles compared to the onco A., DXR, A. oncocalyx 1.2 and onco A 1 increased

(P<0.05) the percentage of TUNEL positive follicles. DXR decreased (P<0.05) the

number of nucleolar organizer regions. Conclusion: A. oncocalyx and onco A affected in

vitro caprine folliculogenesis in a concentration-dependent manner. Onco A (1 µg/ml)

has a less harmful effect than DXR on goat preantral follicle survival.

Keywords: Auxemma oncocalyx; Boraginaceae; in vitro culture; Oncocalyxone A;

Folliculogenesis; Cortical tissue.

63

1. Introduction

Cancer is a target of research for the development or discovery of new forms of

treatments [1]. Many drugs used in cancer chemotherapy, in addition to showing toxicity

against tumor cells, exhibit genotoxic, carcinogenic and teratogenic effects on normal

cells. The low specificity of these drugs against tumor tissue results in undesirable effects.

Doxorubicin (DXR) is a widely used drug for cancer patients [2]. It induces ovarian

toxicity by reducing the ovulation rate, along with a reduction in the size of the ovary and

other side effects [2–4].

Plants are excellent sources of raw material when searching for new drugs.

Moreover, plants have a long history of use in the treatment of cancer [5]. Over 60% of

currently used anti-cancer agents are derived from natural sources, including plants,

marine organisms and micro-organisms [6,7].

AuxemmA. oncocalyx (A. oncocalyx) is a common tree found in the state of Ceará

in Northeast Brazil [8]. It has been widely used in folk medicine as an adjunctive

treatment of injuries such as wounds and cuts [8,9]. Some studies have suggested that this

plant has biological activities such as analgesic, anti-oxidant, anti-tumor and anti-

inflammatory effects [9–12]. Oncocalyxone A (onco A) has been isolated from the stem

heartwood of the plant, which has a high antioxidant activity [9] and an antiproliferative

effect in tumor cell cultures [13].

In other studies, onco A has been suggested as a possible anticancer compound

since it has presented antitumor and cytotoxic activity in human leukemia cells, and other

cell cancer lines, without causing genotoxicity [14]. Therefore, this compound may be

presented as a possible therapeutic agent [15,16]. On the other hand, a study conducted

with sea urchin embryos observed a fraction of onco A induced destruction of the

64

embryos at the same concentrations (1 to 30 µg/ml) which inhibited tumor cell

proliferation, indicating that this compound could be very toxic [13]. However, the effect

of the use of these substances with antitumoral potential on fertility and ovarian follicle

function, for example, is still unknown. Toxicity can be evaluated both in vivo and in

vitro. In vitro studies permit testing drug toxicity while avoiding the ethical concerns and

restrictions of in vivo experiments. Within the criteria required to safely study the effects

of phytotherapeutics on reproductive toxicity, one way to evaluate this parameter is by

the utilization of in vitro preantral follicle culture. This method enables the testing of the

beneficial or toxic effects of drugs on ovarian follicles in vitro before their use in

experiments involving live humans or animals [17].

Thus, the aim of this study was to evaluate the effect of the concentration-response

curve of the A. oncocalyx and onco A and determine the minimum toxic concentration on

the survival, growth and development of goat preantral follicles in vitro cultured

(Experiment 1), and to evaluate the effects of the minimum toxic concentration of A.

oncocalyx and onco a on apoptosis and proliferation of goat pre-antral follicles in vitro

cultured (Experiment 2)”.

2. Materials and Methods

2.1. Source of ovaries

Ovaries (n = 10: Experiment 1; n = 6: Experiment 2) from eight adult mixed breed

goats were obtained at a local slaughterhouse. The ovaries were washed in 70% alcohol

for approximately 10 s then twice in minimal essential medium (MEM) supplemented

with 100 µg/ml penicillin and 100 µg/ml streptomycin plus HEPES (MEM-HEPES). The

ovaries were transported to the laboratory in thermo flasks at 4 ºC within 1 h [18].

65

2.2. Isolation of onco A from A. oncocalyx

Auxemma oncocalyx was collected in August 2012 at Acarape, state of

Ceará, which is located in Northeast Brazil. The plant was identified by Dr. Maria Iracema

B. Loiola of the Department of Biology of the Universidade Federal do Ceará. A voucher

specimen (No. 18459) has been deposited in the Herbarium Prisco Bezerra (EAC),

Universidade Federal do Ceará.

The air-dried and powdered heartwood (2.5 kg) of A. oncocalyx was extracted

with EtOH (2 x each) at room temperature. The combined extracts were evaporated under

reduced pressure to yield the crude extract (100 g), which was fractionated over silica gel

and eluted with CH2Cl2, CH2Cl2/ EtOAc (7:3 and 1:1), EtOAc, EtOAc/ MeOH (9.5:0,5

and 1:1) to yield after solvent evaporation the correspondent fractions: 6.60, 5.99, 8.01,

3.36, 50.08 and 25.96 g, respectively. The fraction EtOAc/MeOH 9.5:0.5 (50.0 g) was

subjected to a silica gel (200 g) chromatography column using CH2Cl2/ EtOAc 1:1 (200

mL), 7:3 (500 mL), EtOAc (1000 mL) and EtOAc/ MeOH 9.5:0.5 (400 mL) to afford 60

fractions of approximately 30 mL. After comparative analysis by TLC, these fractions

were pooled into 3 main fractions: F1(1-20; 8.2 g), F2(21-54; 25.2 g) and F3(55-60; 16.7

g). F2(21-54; 25.2 g) was subjected twice to chromatography over silica gel eluted with

CH2Cl2/EtOAc 1:1, 7:3, EtOAc, EtOAc/MeOH 9.5:0.5 and MeOH. Fractions

CH2Cl2/EtOAc 7:3 and EtOAc furnished a dark solid which was purified by addition of

acetone followed by filtration. This material (5.5 g), a deep red powder, mp 207–208o

was identified as rel-8a-hydroxy-5-hydroxymethyl-2-methoxy-8ab-methyl-7,8,8a,9-

tetrahydro-1,4-anthracenedione), named oncocalyxone A, as previously described by

Pessoa et al., 1993.

66

1H NMR (200 MHz, DMSO-d6): 6.00 (s, H-3), 6.03 (br d, H-6), 2.52 (br d, J 17.2 Hz, H-

7eq), 2.60 (dd, J 17.2, 3.9 Hz, H-7ax), 3.57 (br s, H-8), 2.90 (d, J 18.4 Hz, H-9ax), 2.34

(d, J 18.4 Hz, H-9eq), 6.50 (s, H-10), 4.16 (br s, 2H-11), 0.74 (s, 3H-12), 3.78 (s, OMe).

13C NMR (50.3 MHz, DMSO-d6): 181.2 (C-1), 159.8 (C-2), 106.4 (C-3), 186.1 (C-4),

134.6 (C-4a), 146.7 (C-5), 128.4 (C-6), 32.0 (C-7), 70.1 (C-8), 38.9 (C-8a), 29.2 (C-9),

133.0 (C-9a), 111.8 (C-10), 135.5 (C-10a), 61.6 (C-11), 21.3 (C-12), 56.7 (OMe).

2.3. In vitro culture of goat ovarian tissue

Ovarian tissue samples from each ovarian pair were cut into fragments

approximately 3 x 3 x 1 mm using a needle and scalpel under sterile conditions. One

fragment (non-cultured control) was immediately fixed in Carnoy’s solution for 12 h for

histological studies. The other fragments of ovarian cortex were transferred to 24-well

culture dishes containing 1 ml of culture medium. In vitro culture was performed at 39

°C in 5% CO2 in a humidified incubator and all media were incubated for 2 h prior to use.

The basic culture medium (cultured control) consisted of α-MEM (pH 7.2 – 7.4)

supplemented with 10 ng/ml of insulin, 5.5 µg/ml transferrin, 5 ng/ml selenium, 2 mM

glutamine, 2 mM hipoxanthine and 1.25 mg/ml bovine serum albumin (BSA) and was

called α-MEM+. The culture medium was replaced every 2 days with fresh medium. All

chemicals used in the present study were purchased from Sigma Chemical Co. (St. Louis,

MO, USA) unless otherwise indicated.

2.4. Experimental design

For Experiment 1, the fragments were randomly divided into 11 groups according

to the following treatments: non-cultured control, α-MEM+ alone (cultured control) or

supplemented with: human recombinant bone morphogenetic protein 15 (BMP-15) at 100

67

ng/ml (R&D Systems; Minneapolis, MN, USA); Dimethyl sulfoxide (DMSO) at 20%

v/v; Doxorubicin (DXR) at 0.3 g/ml; Auxemma oncocalyx (A. oncocalyx) at 1.2, 12 or

34 g/ml; or Oncocalixone A (onco A) at 1, 10 or 30 g/ml. The fraction of A. oncocalyx

contains 80% of onco A [12], therefore in each concentration of A. oncocalyx there was

an equal proportion of onco A. A. oncocalyx and onco A were diluted with DMSO as a

vehicle. Each treatment was repeated five times using the ovaries of five different

animals. The BMP-15 concentration was chosen based on previous studies conducted in

our laboratory [19].

Based on the results from Experiment 1, immunohistochemistry and cell

proliferation analysis were performed in Experiment 2. The ovarian fragments were

randomly divided into 7 groups according to the following treatments: non-cultured

control, α-MEM+ alone (cultured control) or supplemented with BMP-15 (100 ng/ml),

DMSO (20% v/v), DXR (0.3 g/ml), A. oncocalyx (1.2 g/ml) or onco A (1 g/ml). Each

treatment was repeated three times.

2.5. Morphological analysis and assessment of in vitro follicular growth

To evaluate the morphology of caprine follicles in the non-cultured control or after

one or seven days of culture, the tissue fragments were fixated and dehydrated in a graded

series of ethanol, clarified with xylene and embedded in paraffin wax. For each piece of

ovarian cortex, 7 µm sections were mounted on slides, stained with periodic acid Schiff

and hematoxylin, and examined by light microscopy at 400× magnification (Nikon

Eclipse E200).

The follicles were classified as primordial (one layer of flattened granulosa cells

around the oocyte) and growing, i.e. transitional (one layer of flattened granulosa cells

and at least 3 cuboidal granulosa cells around the oocyte), primary (a complete layer of

68

cuboidal granulosa cells around the oocyte) or secondary (oocyte surrounded by two or

more layers of cuboidal granulosa cells). Degenerated follicles were defined as those with

a retracted oocyte pyknotic nucleus and/or were surrounded by disorganized granulosa

cells which were detached from the basement membrane. To evaluate follicular

activation and growth, only intact follicles with a visible oocyte nucleus were recorded

and the proportion of primordial and growing follicles were calculated on day 0 (non-

cultured control) and after one or seven days of culture in all tested treatments.

Follicle and oocyte diameter were recorded. Two perpendicular diameters were

recorded from edge to edge, of the follicle or oocyte, and the average of these two values

was reported as follicle and oocyte diameter, respectively. Each follicle was examined in

every section in which it appeared and matched with the same follicle on adjacent

sections to avoid double counting, thus ensuring that each follicle was only counted once,

regardless of its size.

2.6. Assessment of apoptosis by TUNEL assay

For determination of DNA fragmentation, a terminal deoxynucleotidyl

transferase-mediated dUTP biotin nick end labeling (TUNEL) in situ detection kit (R&D

Systems, Minneapolis, MN, USA) was applied. The fragments were fixed in 4%

paraformaldehyde buffered with PBS. Subsequently, the blocks were sectioned at a

thickness of 5 µm following de-paraffinization and boiled for antigen retrieval in 0.01 M

citric acid. The blockade of exogenous peroxidase and nonspecific blocking were

performed in a humid chamber. The TUNEL kit was prepared following the guidelines

given by the manufacturer. The incubation of TUNEL consisted of the addition of a

TUNEL mixture for 1 h at 37 °C (moist chamber) and, after washing, 50 ml Convert POD

was added for 30 min at 37 °C (moist chamber). The staining of the nucleus of apoptotic

69

cells appeared brown whereas normal cells were lighter in color. At least 30 follicles per

treatment were evaluated.

2.7. Assessment of cell proliferation

2.7.1. AgNOR silver staining

To estimate the cell proliferation index AgNOR staining was performed to

quantify the number of argyrophilic nucleolar organizer regions (NOR). For this purpose,

ovarian tissue fixed in 4% paraformaldehyde solution was sectioned at 5 µm. After

reduction with 1% potassium iodide, slides were stained with 50% silver nitrate solution

in a colloid solution (2:1) in a darkroom and counterstained with 0.1% safranin. For

quantification, the follicles were visualized under a light microscope (1000X

magnification; Nikon Eclipse E200) and the NOR of all the nuclei of all the visible

granulosa cells were counted. In the non-cultured group and groups after 7 days of culture,

granulosa cells from 30 growing preantral follicles were evaluated per group.

2.7.2. Proliferating cell nuclear antigen (PCNA)

Ovarian fragments were fixed in 4% paraformaldehyde and 5 µm paraffin sections

were mounted to microscope slides. Paraffin sections were heated at 65 °C for 45 min.

Following de-paraffinization, sections were rehydrated in a series of graded ethanol/water

solutions then boiled in 0.01 M citric acid (pH 6.0) at 95–100 °C for 5 min followed by

incubation in 3% hydrogen peroxide (H2O2) for 10 min. The tissues were blocked with

avidin and biotin and incubated with a Rb Pab-PCNA ab 2426 (abcam) overnight at 4 °C.

After rinsing thoroughly with PBS, the sections were incubated with goat pAB-Rb IgG

antibody (Biotin) for 30 min at room temperature. PCNA expression in sections was

detected by the reaction of peroxidase with 3,39-diaminobenzidine tetrahydrochloride

70

(DAB) and analyzed using a light microscope (400X maginification, Nikon Eclipse

E200). When at least one granullosa cell was marked in brown, it was considered as a

positive PCNA follicle. At least 20 follicles per treatment were evaluated.

2.8. Statistical analyses

Data were initially evaluated for homocedasticity and normal distribution of the

residues by Bartlett’s and Shapiro-Wilk tests, respectively. Confirmed both requirements

underlying analysis of variance, the effects of medium, time and medium by time

interaction were analyzed using PROC MIXED of SAS (2002), including repeated

statement to account for autocorrelation between sequential measurements. The model

was Yij=µ+Mi+Tj+(R*T)ij+eij, where Yij is the observation of the ith medium at the jth

time of culture, µ is the overall mean, Ri is the ith medium, Tj is the jth time of culture,

(R*T)ij is the medium by time interaction term and eij is the random residual effect.

Comparisons amongst media or times were further analyzed by Student-Newman-Keuls

test, being the results expressed as mean ± standard deviation. TUNEL and PCNA data

were analyzed by chi-square test and the results were expressed as percentages. A

probability of P<0.05 indicated a significant difference.

3. Results

3.1. Morphologically normal preantral follicles and follicle and oocyte diameter before

and after in vitro culture

A total of 3,150 preantral follicles were analyzed by classical histology. The

percentage of morphologically normal preantral follicles in the non-cultured control

treatment and after 1 or 7 days of in vitro culture is shown (Table 1). After 1 and 7 days

71

of culture there was a reduction (P<0.05) in the percentage of normal follicles in all

treatments compared to the non-cultured control. When compared to α-MEM+, the

percentage of morphological normal follicles was higher (P<0.05) on days 1 and 7 in the

BMP-15 treatment. However, the percentage of morphological normal follicles was

similar (P>0.05) between the DMSO and α-MEM+ treatments. On the other hand, the

treatments with DXR, A. oncocalyx and onco A at all concentrations were lower (P<0.05)

than α-MEM+ on days 1 and 7. The A. oncocalyx 1.2 (day 1), onco A 1 (days 1 and 7) and

onco A 10 (day 1) treatments showed a higher (P<0.05) percentage of morphologically

normal follicles than DXR. On day 1, the treatments with A. oncocalyx 12 and 34, and on

day 7, those with A. oncocalyx 12 and 34 and onco A 30 showed a lower (P<0.05)

percentage of morphologically normal follicles compared to the DXR treatment.

Comparing the concentration of A. oncocalyx with its equivalent of onco A, there was a

lower percentage of normal follicles in the A. oncocalyx treatments. Among the different

concentrations of A. oncocalyx and onco A, it was observed that after 1 and 7 days of

culture there was a concentration-dependent effect with a decrease in the percentage of

normal follicles along with an increase in A. oncocalyx or onco A concentrations. Also,

with the progression of the culture from day 1 to day 7, there was a reduction (P<0.05) in

the percentage of normal follicles in all treatments. Finally, there was no difference

(P>0.05) in follicle and oocyte diameter among treatments (data not shown).

3.2. Activation of caprine primordial follicles after in vitro culture

The percentages of preantral follicle activation in the non-cultured control and

after 1 or 7 days of in vitro culture are shown (Table 2). After 7 days of culture, a lower

(P<0.05) percentage of primordial follicles and an increase (P<0.05) in the percentage of

growing follicles was observed in all treatments compared to the non-cultured control,

72

except the DMSO treatment. From day 1 to day 7, a significant reduction in the percentage

of primordial follicles and a significant increase in the percentage of growing follicles

were observed in all treatments. Overall, irrespective of culture period, the

supplementation of α-MEM+ with DMSO, BMP-15, DXR, A. oncocalyx and onco A did

not significantly affect either the percentage of primordial or growing follicles.

3.3 TUNEL assay

The percentage of TUNEL positive follicles (TPF; Fig. 1) was similar (P>0.05) in

the α-MEM+, DMSO and BMP-15 treatments compared to the non-cultured control. In

contrast, DXR, A. oncocalyx 1.2 and onco A 1 showed a higher (P<0.05) percentage of

TPF compared to the non-cultured control, DMSO and BMP-15 treatments. Moreover,

the addition of onco A 1 did not increase (P>0.05) the percentage of TPF compared to the

α-MEM+ treatment.

3.4. Assessment of proliferation with AgNOR and PCNA

The mean number of nucleolar organizer regions (NOR) per treatment is shown

(Fig. 2). DXR was the only treatment that had a lower (P<0.05) number of NOR than the

non-cultured control, α-MEM+, BMP-15 and onco A 1 treatments. It is important to

emphasize that BMP-15 was the only treatment that increased (P<0.05) the percentage of

NOR compared to the α-MEM+ treatment. On the other hand, the PCNA test showed no

statistical difference (P>0.05) among all treatments (Fig. 3).

73

4. Discussion

The present study demonstrated for the first time the effect of A. oncocalyx

and its isolated compound, onco A, on ovarian preantral folicles. After 7 days of culture,

a concentration-dependent effect was observed of both A. oncocalyx and onco A, of which

a higher concentration was related to a smaller percentage of morphologically normal

follicles when compared to the α-MEM+ cultured control. However, both compounds did

not affect either the percentage of primordial or growing follicles, showing that the effect

of this drugs is not stage-specific, and they cause the degeneration of both categories

equally, as it has been seen with other toxic compounds such as Areca catechu [20]. In a

study testing different concentrations (1 to 100 µg/ml) of a quinone fraction of A.

oncocalyx (containing 80% of onco A) in sea urchin eggs, it was found that the cleavage

of eggs was inhibited in a concentration-dependent manner. The early destruction of

embryos at the blastula stage occurred when a concentration of 10 µg/ml was used, and a

total destruction (100%) of embryos occurred with a concentration of 30 µg/ml,

associated with a rupture of the embryo membranes [21]. The cytotoxicity of onco A was

also observed on human tumor cell lines [15,16,22] and normal skin fibroblasts [22] at

concentrations varying from 0.4 to 25 µg/ml. In addition, onco A inhibited leukemia cell

line CEM growth at concentrations greater than 2 µg/ml and showed a significant

deleterious effect on DNA [15,16,22]. The cytotoxicity associated with these compounds

can be attributed to redox cycling and subsequent development of oxidative stress [23].

Thus, cellular damage can occur by DNA alkylation [24]. Consequently, quinones might

have a high toxicity related to their antimitotic properties [15].

In the present study, the fraction of A. oncocalyx had a higher toxicity than onco

A on folliculogenesis. Several studies [14–16,21,22] have already shown in vitro

74

cytotoxic effects of the quinone fraction of A. oncocalyx, represented mainly by onco A.

However, Ferreira et al. [9] showed an antioxidant effect of the fraction of A. oncocalyx

on mice in vivo. It is believed that this effect can be due to prevention of the process of

lipid peroxidation and enhancement of protein synthesis [9]. Moreover, the fraction of A.

oncocalyx contains 80% of onco A [12], leaving the other 20% of other substances that

can be more toxic than onco A alone. Pessoa et al. [25] listed some compounds of this

fraction, such as cordiachromes, allantoin, sitosterol, 3b-O-b-D-glucopyranosylsitosterol

and acetyl derivative. It is known that the cordiachromes are an unusual class of

meroterpenoids that have been isolated from a quinone [26]. Cordiachromes were also

isolated from the trunk heartwood of another Northeastern Brazilian tree (Auxemma

glazioviana; [27]). The wood of this tree, as is that of A. oncocalyx, is resistant to fungi

and termite attacks, and thus is often used for civil construction [25,27]. In folk medicine,

cuts and wounds were treated with the trunk bark [8,25]. It has been suggested that this

property can be related to the cordiachromes present in the quinone fraction [26]. These

properties can influence the toxicity of the plant and may explain the difference between

the fraction of A. oncocalyx and onco A.

The results showed that onco A was less harmful than DXR on follicle survival

as well as the proliferation of granulosa cells. It is known that onco A and DXR both have

anti-carcinogenic effects. The fraction of A. oncocalyx and onco A in different lineages

of cells, especially in tumor cell lines [14,16,22], showed a similar effect when compared

to DXR. It was shown that both DXR and onco A present cytotoxicity in the G, G1/S and

S phases of cell division, with the only difference being that DXR presents genotoxicity

in these cells [14]. In this study, DXR caused a decrease in both the percentage of

morphologically normal follicles and the proliferation of granulosa cells and also

increased follicular DNA fragmentation. Likewise, the TUNEL assay showed an increase

75

in apoptotic cells in the groups treated with the A. oncocalyx 1.2 and onco A 1 treatments

when compared to the non-cultured control. Only the onco A 1 treatment was able to

maintain a percentage of TUNEL positive follicles similar to the α-MEM+ cultured

control. A study conducted by Pessoa et al. [14] showed that onco A at a concentration

of 0.5 g/ml had a similar effect as 0.1 mg/ml DXR with regard to cytotoxicity in

lymphocytes. Many drugs used in cancer chemotherapy, in addition to having toxicity

against tumor cells, exhibit genotoxic, carcinogenic and teratogenic effects on normal

cells, showing a low specificity of these drugs against tumor tissue, resulting in

undesirable side effects. Nevertheless, DXR is a widely used drug in cancer treatment

[28]. Recent studies revealed that DXR induced ovarian toxicity, which was observed by

a reduction in ovulation rate and the size of the ovary [2–4]. DXR elicits apoptosis by

various mechanisms in a variety of cells. It can be accumulated in both the nucleus and

mitochondria and induces chromosomal destruction by inhibiting topoisomerase-II [29].

DXR can also interfere with mitochondrial function and initiate an intrinsic pathway of

apoptosis by reducing the mitochondrial membrane potential and releasing cytochrome

C [3].

It is important to highlight that the DMSO treatment (i.e., the vehicle used for the

dilution of DXR, A. oncocalyx and onco A) was similar to the α-MEM+ cultured control.

This result shows that DMSO by itself was not responsible for the negative effect of the

tested drugs. On the other hand, in this study BMP-15 served as a positive control for its

ability to maintain follicular viability, increase granulosa cell proliferation and prevent

DNA fragmentation. Celestino et al. [19] showed that BMP-15 (100 ng/ml) maintained

the integrity and promoted the growth of caprine preantral follicles cultured in vitro for 7

days. It is known that BMP-15, along with the other BMPs (2 and 5), acts in granulosa

76

cells, promoting follicle survival through maintenance of cell proliferation and prevention

of precocious luteinization and/or atresia [30].

In conclusion, A. oncocalyx and onco A affected caprine folliculogenesis in vitro

in a concentration-dependent manner. In addition, the less harmful effect of onco A (1

µg/ml) than DXR on goat preantral follicle survival may encourage future studies

involving the use of this drug for cancer treatment in women.

5. Conflict of interest




6. Acknowledgments

This research was financially supported by CNPq, CAPES and FUNCAP. The

authors thank Denise Damasceno Guerreiro, Naiza de Sá Arcangela, Renato Felix da

Silva, Francisco Léo Nascimento de Aguiar and Keith Haag for assistance with this study.

79

Table 1. Percentage (mean ± SEM) of morphologically normal caprine preantral follicles

before (non-cultured control) and after in vitro culture for 1 or 7 days in α-MEM+ alone

or α-MEM+ supplemented with DMSO, BMP15, DXR, A. oncocalyx (1.2; 12 and 34

µg/ml) or onco A (1, 10 and 30 µg/ml).

Treatments Culture time

Non-cultured control 88.00 ± 1.83

D1 D7

α-MEM+ cultured control 74.67 ± 2.98 *A 54.67 ± 1.83 *B

BMP-15 82.00 ± 5.06 *†Aa 69.79 ± 2.40 *†Ba

DMSO 71.33 ± 3.80 *Ab 54.00 ± 4.35 *Bb

DXR 45.33 ± 5.06 *†Ae 32.67 ± 5.48 *†Bd

A oncocalyx 1.2 55.33 ± 1.83 *†Ad 27.33 ± 4.35 *†Bde

A oncocalyx 12 34.67 ± 3.80 *†Af 20.67 ± 3.65 *†Bf

A oncocalyx 34 24.00 ± 1.49 *†Ag 14.00 ± 2.79 *†Bg

Onco A 1 64.00 ± 4.35 *†Ac 48.00 ± 5.06 *†Bc

Onco A 10 58.67 ± 6.06 *†Ad 32.67 ± 6.41*†Bd

Onco A 30 42.67 ± 5.48 *†Ae 24.67 ± 3.80 *†Bef

* Differs significantly from non-cultured control. † Differs significantly from α-MEM+.

Distinct capital letters represent significant differences between columns (days of

culture). Different lowercase letters represent significant differences between lines

(experimental treatments).

80

Table 2. Percentage (mean ± SEM) of primordial and growing caprine preantral follicles before (non-cultured control) and after in vitro

culture for 1 or 7 days in α-MEM+ alone or α-MEM+ supplemented with DMSO, BMP15, DXR, A. oncocalyx (1.2; 12 and 34 µg/ml) or onco

A (1, 10 and 30 µg/ml).

Treatments Primordial follicles Growing follicles

Culture Time

Non-cultured

control

65.95 ± 4.92 34.05 ± 4.92

D1 D7 D1 D7

α-MEM+ cultured

control

56.25 ± 4.64 A 19.56 ± 5.18*B 43.75 ± 4.64 B 80.44 ± 5.18*A

BMP-15 41.72 ± 7.86*Ab 9.10 ± 3.80*Bb 58.28 ± 7.86*Ba 90.90 ± 3.80*Aa

DMSO 59.14 ± 8.78 Aa 22.19 ± 10.67*Bab 40.86 ± 8.78Bb 77.81 ± 10.67*Aab

DXR 40.38 ± 18.95*Ab 21.69 ± 7.63*Bab 59.62 ± 18.95*Ba 78.31 ± 7.63*Aab

A oncocalyx 1.2 49.41 ± 5.06 *Aab 14.44 ± 3.38*Bab 50.59 ± 5.06*Bab 85.56 ± 3.38*Aab

A oncocalyx 12 38.65 ± 6.80*†Aab 22.29 ± 6.19*Bab 61.35 ± 6.80*†Ba 77.71 ± 6.19*Aab

A oncocalyx 34 47.14 ± 6.39*Aab 28.67 ± 7.94*Ba 52.86 ± 6.39*Bab 71.33 ± 7.94*Ab

Onco A 1 51.90 ± 5.63*Aab 23.96 ± 9.85*Bab 48.10 ± 5.63*Bab 76.04 ± 9.85*Aab

Onco A 10 49.19 ± 6.07*Aab 18.72 ± 8.78*Bab 50.81 ± 6.07*Bab 81.28 ± 8.78*Aab

Onco A 30 37.56 ± 4.59*†Ab 24.48 ± 10.52*Bab 62.44 ± 4.59*†Ba 75.52 ± 10.52*Aab

* Differs significantly from non-cultured control. † Differs significantly from α-MEM+ cultured control. Distinct capital letters represent

significant differences between columns (days of culture). Different lowercase letters represent significant differences between lines

(experimental treatments).

81

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8 EFEITO DA TOXICIDADE DA FRAÇÃO DA AUXEMMA ONCOCALYX E

DO PRINCÍPIO ATIVO ONCOCALYXONE A NO CULTIVO IN VITRO DE

FOLÍCULOS SECUNDÁRIOS E NA MATURAÇÃO IN VITRO DE OÓCITOS

DE CAPRINOS

“Toxicity effect of the Auxemma oncocalyx fraction and its active principle

oncocalyxone A on in vitro culture of caprine secondary follicles and in vitro oocyte

maturation.”

Periódico: Semina: Ciências Agrárias (aceito) (ISSN: 1679-0359)

Qualis B1

85

RESUMO

O extrato da Auxemma oncocalyx (A. oncocalyx) e seu componente, oncocalyxona A

(onco A), possui atividade antitumoral, podendo afetar a fertilidade. Entretanto, estudos

sobre a ação dessas substâncias em relação à foliculogênese caprina são desconhecidos.

O objetivo desse estudo foi avaliar o efeito da A. oncocalyx e onco A no cultivo in vitro de

folículos secundários isolados (Experimento 1) e na maturação in vitro (MIV) de oócitos

de folículos antrais caprinos crescidos in vivo (Experimento 2). Folículos secundários

isolados foram distribuídos em seis grupos, em que o controle não-cultivado foi

imediatamente fixado em paraformaldeído 4%. Os folículos restantes foram cultivados

durante 7 dias em α-MEM+ sozinho (controle) ou suplementado com DMSO,

doxorrubicina (DXR), A. oncocalyx ou onco A. Após o cultivo, os folículos foram

avaliados quanto à formação de antro, taxa de crescimento, apoptose (TUNEL) e

proliferação celular (PCNA), bem como a expressão dos genes BCL2 e BAX. Além disso,

os complexos cumulus-oócitos (CCOs) foram aspirados e distribuídos em cinco

tratamentos para MIV: o controle em meio de maturação (TCM 199+), e os demais

tratamentos suplementados com DMSO, DXR, A. oncocalyx ou onco A. Depois da MIV,

a configuração da cromatina e viabilidade oocitária foram avaliadas. Após 7 dias de

cultivo, observou-se redução na percentagem de folículos morfologicamente intactos, na

formação de antro, na taxa de crescimento e no número de células PCNA positivas

(P<0,05). Depois do cultivo, no tratamento DXR foi observada maior percentagem de

folículos TUNEL positivos (P<0,05) e também aumento na taxa de RNAm BAX: BCL2

(P<0,05). Após MIV dos CCOs, nos tratamentos com DXR, A. oncocalyx e onco A,

observou-se maior (P<0,05) percentagem de oócitos anormais e menor de oócitos viáveis

quando comparados ao grupo controle (P<0,05). No entanto, apenas nos tratamentos

DXR e onco A aumentou a percentagem de oócitos viáveis com configuração da

cromatina anormais (P<0,05). Não houve diferenças nas taxas de maturação entre o grupo

controle e os tratamentos DXR, A. oncocalyx e onco A. De acordo com nossas condições

de cultivo, pode-se concluir que a A. oncocalyx e onco A não apresentaram efeitos tóxicos

sobre folículos secundários isolados e as taxas de maturação dos CCOs recuperados a

partir de folículos antrais. No entanto, estas substâncias afetam negativamente a

viabilidade oocitária. Assim, o uso de biotecnologias como o cultivo de folículos

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secundários in vitro e MIV de oócitos para testes de toxicidade são métodos apropriados

para avaliar possíveis efeitos das drogas na foliculogênese.

Palavras-chave: Auxemma oncocalyx. Doxorrubicina. Oncocalyxona A. Foliculogênese.

Ovócitos

87

Efeito da toxicidade da fração da Auxemma oncocalyx e do princípio ativo oncalyxone A

no cultivo in vitro de folículos secundários e na maturação in vitro de oócitos de caprinos.

Toxicity effect of the Auxemma oncocalyx fraction and active principle oncalyxone A

on in vitro culture of caprine secondary follicles and in vitro oocyte maturation.

Leiva-Revilla J.a,, Cadenas J.a, Vieira L. a, Macedo V. a, Campello C.C. a, Aguiar F.L. a,

Celestino J.J.H. b, Pessoa O.D.L. c, Apgar G.A. d, Rodrigues A.P.R. a, Figueiredo J.R. a*,

Maside C. a.

a Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA),

Universidade Estadual do Ceará, Fortaleza-CE, Brazil.

b Institute of Health Sciences, Universidade da Integração Internacional da Lusofonia

Afro-Brasileira. Acarape-CE, Brazil.

c Departamento de Química Orgânica e Inorgânica. Universidade Federal do Ceará,

Centro de Ciências. Fortaleza-CE, Brazil

d Professor of Department of Animal Science, Food and Nutrition, Southern Illinois

University-Carbondale, USA

* Correspondence should be addressed to:

Laboratório de Manipulação de Oócitos e Folículos Pré-Antrais (LAMOFOPA).

Universidade Estadual do Ceará (UECE). Av. Silas Munguba, 1700, Campus do Itaperi.

Fortaleza – CE – Brasil. CEP: 60740 903

Tel.: +55.85. 3101.9852; Fax: +55.85.3101.9840

E-mail address: [email protected]

88

ABSTRACT

The extract of Auxemma oncocalyx (A. oncocalyx) and its main component i.e.,

oncocalyxone A (onco A), have anti-tumoral activity, and might affect fertility. Studies

on the action of these substances regarding caprine folliculogenesis are lacking. The aim

of this study was to evaluate the effect of A. oncocalyx and onco A on the in vitro culture

of isolated secondary follicles (Experiment 1) and on the in vitro maturation (IVM)

(Experiment 2) of oocytes from caprine antral follicles grown in vivo. Isolated secondary

follicles were distributed in six groups; the non-cultured control was immediately fixed

in Paraformaldehyde 4%. The remaining follicles were cultured for 7 days in α-

MEM+ alone (control) or supplemented with DMSO, doxorubicin (DXR), A.

oncocalyx or onco A. After culture, follicles were evaluated for antrum formation, growth

rate, apoptosis (TUNEL) and cellular proliferation (PCNA), as well as gene expression

of BCL2 and BAX. Additionally, cumulus oocyte complexes (COCs) were aspirated and

allocated into five treatments for IVM: control, cultured only in maturation base medium

(TCM 199+); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After IVM,

oocyte chromatin configuration and viability were assessed. After 7 days of culture, there

was a reduction in the percentage of morphologically intact follicles, antrum formation,

growth rate and number of PCNA positive granulosa cells (P < 0.05). After culture, the

DXR treatment had a higher percentage of TUNEL positive follicles and relative

BAX:BCL2 mRNA ratio’s (P < 0.05). After IVM of the COCs, DXR, A. oncocalyx and

onco A treatments had a greater percentage (P < 0.05) of abnormal oocytes and a lower

percentage of viable oocytes as compared with the control group (P < 0.05). However,

only DXR and onco A treatments increased the percentage of alive oocytes with abnormal

chromatin configuration (P < 0.05). There were no differences in maturation rates

between the control group and DXR, A. oncocalyx and onco A treatments. In conclusion,

under our culture conditions, A. oncocalyx and onco A do not exhibit a toxic effect on

isolated secondary follicles and on maturation rates of COCs recovered from antral

follicles. However, these substances negatively affected the oocyte viability. Thus, the

use of culture biotechnologies as an in vitro secondary follicle culture and in vitro oocyte

maturation toxicity testing are appropriated methods to evaluate the possible effects of

drugs in folliculogenesis.

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Keywords: Auxemma oncocalyx; Doxorubicin; Oncocalyxone A; Folliculogenesis;

Oocytes.

90

1. Introduction

In vitro manipulation of oocytes enclosed in preantral follicles allows drug

toxicity testing, by avoiding the ethical concerns and restrictions of in vivo experiments.

Therefore, one way to safely study the effects of drugs on reproduction, is by the

utilization of in vitro preantral follicle culture. This technique enables the testing of

beneficial or toxic effects on ovarian follicles in vitro before their use in experiments

involving live subjects (FIGUEIREDO et al., 2011).

Cancer research is continuously seeking to develop or discover new treatments

(CRAGG et al., 2014). Many drugs used in cancer chemotherapy, in addition to showing

toxicity against tumor cells, exhibit genotoxic, carcinogenic and teratogenic effects on

normal cells. One of the most commonly used is Doxorubicin (DXR) (OKTEM; OKTAY,

2007). Normally used in treatment against bladder, breast, lung, ovary cancer and others

(CHOW et al., 2010), it transgresses the cell membrane and accumulates in both the

nucleus mitochondria, by inducing oxidative stress and chromosomal obliteration through

inhibition of topoisomerase II (MAILER; PETIRING, 1976). However, DXR induces

ovarian toxicity by reducing the ovulation rate, along with a reduction in the size of the

ovary and other side effects (OKTEM; OKTAY, 2007; BAR-JOSEPH et al., 2010; BEN-

AHARON et al., 2010). The poor specificity of these drugs against tumor tissue highlights

the need of developing new drugs with fewer side effects (OKTEM; OKTAY, 2007) and

more specificity.

Plants are excellent sources of raw material when searching for new drugs, and

commonly used in the treatment of cancer (GRAHAM et al., 2000). In addition, over 60%

of currently anti-cancer agents are derived from natural sources, including plants, marine

organisms and micro-organisms (NEWMAN et. al., 2003; CRAGG; NEWMAN, 2005).

Auxemma oncocalyx (A. oncocalyx) is a common tree found in the state of Ceará in

Northeast Brazil (BRAGA, 1976). It has been widely used in folk medicine as an

adjunctive treatment for injuries (BRAGA, 1976; FERREIRA et al., 2003). Some studies

have suggested that this plant has biological activities such as analgesic, anti-oxidant,

anti-tumor and anti-inflammatory effects (PESSOA et al., 1992; LINO et al., 1996;

FERREIRA et al., 2003, 2004). Oncocalyxone A (onco A) has been isolated from the

stem heartwood of the plant, which has a high antioxidant activity (FERREIRA et al.,

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2003) and an antiproliferative effects in tumor cell cultures (COSTA-LOTUFO et al.,

2002).

In previous studies, onco A has been suggested as a possible anticancer compound

since it has presented antitumor and cytotoxic activity in human leukemia cells, and other

cell cancer lines, without causing genotoxicity (LEYVA et al., 2000; PESSOA et al.,

2003, 2004). However, the effect of A. oncocalyx and onco A on in vitro folliculogenesis

is not known.

Hence, the aim of this study was to investigate the effect of A. oncocalyx and onco

A on the in vitro survival and growth of isolated goat secondary follicles (Experiment 1)

as well as on the viability and nuclear maturation of oocytes recovered from antral

follicles (Experiment 2).

2. Materials and methods

2.1. Source of ovaries

Ovaries (n =130) from 65 cycled mixed breed adult goats, with body condition

score 3 (30 and 35 for experiment 1 and 2, respectively) were obtained at a local

slaughterhouse located in Mossoro, Rio Grande do Norte state, northeast region, with

latitude: 05º 11' 15" S and longitude 37º 20' 39" W. The ovaries were washed in 70%

alcohol for approximately 10 s and then twice in minimal essential medium (MEM)

supplemented with 100 µg/mL penicillin and 100 µg/mL streptomycin plus HEPES

(MEM-HEPES). The ovaries were transported to the laboratory at 4 or 33 °C (experiment

1 or 2, respectively) in a thermal container, within 4 h. All chemicals used in the present

study were purchased from Sigma Chemical Co. (St. Louis, MO, USA), unless otherwise

indicated.

2.2. Obtainment of onco A from A. oncocalyx

Auxemma oncocalyx was collected in August 2012 at Acarape, state of Ceará,

which is located in Northeast Brazil. The plant was identified by Dr. Maria Iracema B.

Loiola of the Department of Biology of Federal University of Ceará. A voucher specimen

(No. 18459) has been deposited in the Herbarium Prisco Bezerra (EAC), Federal

University of Ceará.

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The air-dried and powdered heartwood (2.5 kg) of A. oncocalyx was extracted

with EtOH (2 x each) at room temperature. The combined extracts were evaporated under

reduced pressure to yield the crude extract (100 g), which was fractionated over silica gel

and eluted with CH2Cl2, CH2Cl2/ EtOAc (7:3 and 1:1), EtOAc, EtOAc/ MeOH (9.5:0.5

and 1:1) to yield after solvent evaporation the correspondent fractions: 6.60, 5.99, 8.01,

3.36, 50.08 and 25.96 g, respectively. The fraction EtOAc/MeOH 9.5:0.5 (50.0 g) was

subjected to a silica gel (200 g) chromatography column using CH2Cl2/ EtOAc 1:1 (200

mL), 7:3 (500 mL), EtOAc (1000 mL) and EtOAc/ MeOH 9.5:0.5 (400 mL) to afford 60

fractions of approximately 30 mL. After comparative analysis by TLC, these fractions

were pooled into 3 main fractions: F1 (1-20; 8.2 g), F2 (21-54; 25.2 g) and F3 (55-60;

16.7 g). F2 (21-54; 25.2 g) was subjected twice to chromatography over silica gel eluted

with CH2Cl2/EtOAc 1:1, 7:3, EtOAc, EtOAc/MeOH 9.5:0.5 and MeOH. Fractions

CH2Cl2/EtOAc 7:3 and EtOAc furnished a dark solid, which was purified by addition of

acetone followed by filtration. This material (5.5 g), a deep red powder, mp 207–208o

was identified as rel-8a-hydroxy-5-hydroxymethyl-2-methoxy-8ab-methyl-7,8,8a,9-

tetrahydro-1,4-anthracenedione), named onco A, as previously described by PESSOA et

al., 1993.

1H NMR (200 MHz, DMSO-d6): 6.00 (s, H-3), 6.03 (br d, H-6), 2.52 (br d, J 17.2

Hz, H-7eq), 2.60 (dd, J 17.2, 3.9 Hz, H-7ax), 3.57 (br s, H-8), 2.90 (d, J 18.4 Hz, H-9ax),

2.34 (d, J 18.4 Hz, H-9eq), 6.50 (s, H-10), 4.16 (br s, 2H-11), 0.74 (s, 3H-12), 3.78 (s,

OMe). 13C NMR (50.3 MHz, DMSO-d6): 181.2 (C-1), 159.8 (C-2), 106.4 (C-3), 186.1

(C-4), 134.6 (C-4a), 146.7 (C-5), 128.4 (C-6), 32.0 (C-7), 70.1 (C-8), 38.9 (C-8a), 29.2

(C-9), 133.0 (C-9a), 111.8 (C-10), 135.5 (C-10a), 61.6 (C-11), 21.3 (C-12), 56.7 (OMe).

It is noteworthy that the fraction of A. oncocalyx contains 80 % of onco A

(FERREIRA et al., 2004), therefore the concentration of A. oncocalyx was in equal

proportion of onco A. A. oncocalyx and onco A were diluted with DMSO as a vehicle.

The concentrations of A. oncocalyx and onco A were chosen based on previous studies

performed in our laboratory (unpublished data).


For Experiment 1, isolated secondary follicles were randomly distributed in the

following six treatments: I) non-cultured control; II) cultured in α-MEM+ (control); III)

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α-MEM+ supplemented with 20% v/v dimethyl sulfoxide (DMSO); IV) α-MEM+

supplemented with 0.3 g/mL DXR (positive toxicity control); V) α-MEM+

supplemented with 1.2 g/mL A. oncocalyx or VI) α-MEM+ supplemented with 1 g/mL

onco A. The culture was replicated seven times, and a total of 60 follicles (at least) were

used in each treatment. Cultured follicles were evaluated for antrum formation capacity,

growth rate, apoptosis (TUNEL) and cellular proliferation (PCNA). In addition, both non-

cultured and cultured isolated secondary follicles from the five treatments after culture

were selected for Bcl2 and Bax gene expression.

For experiment 2, COCs were allocated into five treatments to perform in vitro

maturation: I) TCM-199+ (control) II) TCM-199+ supplemented with 20% v/v DMSO;

III) TCM-199+ supplemented with 0.3 g/mL DXR; IV) TCM-199+ supplemented with

1.2 g/mL A. oncocalyx or V) TCM-199+ supplemented with 1 g/mL onco A. The

culture (maturation) was replicated three times, and a total of 75 (at least) COCs were

used in each treatment. After 24 h of in vitro maturation, oocyte chromatin configuration

and viability were assessed by fluorescence microscopy.

2.4. Isolation, selection and culture of secondary follicles

After transportation, fat and connective tissue surrounding the ovaries were

removed. Cortical slices (1 to 2 mm thick) were cut with a surgical blade (under sterile

conditions) and placed in a holding medium consisting of HEPES-MEM. Secondary

follicles that were approximately 200 µm in diameter were visualized using a

stereomicroscope (SMZ 645, Nikon, Tokyo, Japan) with ocular micrometer (100 X

magnification) and manually dissected from strips of ovarian cortex using 26-gauge (26

G) needles. After isolation, follicles were transferred to 100 µL drops containing fresh

culture medium under mineral oil for further evaluation of follicular quality. Follicles

with a visible and centrally located oocyte that were surrounded by granulosa cells and

had an intact basement membrane and no antrum formation were selected as secondary

follicles for culture.

After selection, follicles were individually cultured in 100 µL drops of culture

media and allocated into different treatments (67, 58, 59, 59 and 58 respectively for

control, DMSO, DXR, A. oncocalyx and onco A treatments) described further in the

experimental design (item 2.5). The base culture media consisted in α-MEM

94

supplemented with 3 mg/mL bovine serum albumin (BSA), 10 ng/mL insulin, 5.5 µg/mL

transferrin and 5 ng/mL selenium, 2 mM glutamine, 2 mM hypoxanthine, 50 µg/mL

ascorbic acid and 100 ng/mL FSH (α-MEM+). The culture was carried out at 39 °C, in

5% CO2 in air for 7 days. Fresh media were prepared and pre-equilibrated for 2 h prior to

use. Every other day, 60 µL of medium were replenished in each drop.

2.5. Assessment of follicle development

During culture, follicles were classified according to their morphology. Follicles

showing darkness of the oocytes and surrounding cumulus cells or those with misshapen

oocytes were classified as degenerated. At day 7 of culture, follicular diameter and antrum

formation were evaluated only in healthy follicles. The follicular diameter was

determined as the mean of two perpendicular measures of each follicle, using an ocular

micrometer (100 X magnification) inserted into a stereomicroscope (SMZ 645, Nikon,

Tokyo, Japan). Antrum formation was defined as a visible translucent cavity within the

granulosa cell layers.

2.6. Assessment of apoptosis by TUNEL assay

For determination of DNA fragmentation, a terminal deoxynucleotidyl

transferase-mediated dUTP biotin nick end labeling (TUNEL) detection kit (R&D

Systems, Minneapolis, MN, USA) was utilized. The follicles were fixed in 4%

paraformaldehyde buffered with PBS. Subsequently, the blocks were sectioned at a

thickness of 5 µm following de-paraffinization and boiled for antigen retrieval in 0.01 M

citric acid. The blockade of exogenous peroxidase and nonspecific blocking were

performed in a humid chamber. The TUNEL kit was prepared following the guidelines

provided by the manufacturer. The incubation of TUNEL consisted of the addition of a

TUNEL mixture for 1 h at 37 °C (moist chamber) and, after washing, 50 mL Convert

POD was added for 30 min at 37 °C (moist chamber). Follicles were considered TUNEL

positive when oocyte nucleus was stained brown.

2.7. Proliferating cell nuclear antigen (PCNA) assessment

Follicles were fixed in 4% paraformaldehyde and 5 µm paraffin sections were

mounted to microscope slides. Paraffin sections were heated at 65 °C for 45 min.

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Following de-paraffinization, sections were rehydrated in a series of graded ethanol/water

solutions then boiled in 0.01 M citric acid (pH 6.0) at 95–100 °C for 5 min followed by

incubation in 3% hydrogen peroxide (H2O2) for 10 min. The tissues were blocked with

avidin and biotin and incubated with a Rb Pab-PCNA (1:3000 - 2426 Abcam) overnight

at 4 °C. After rinsing thoroughly with PBS, the sections were incubated with caprine

pAB-Rb IgG antibody (Biotin) for 30 min at room temperature. PCNA expression was

detected by the reaction of peroxidase with 3,39-diaminobenzidine tetrahydrochloride

(DAB) and analyzed using a light microscope (400X maginification, Nikon Eclipse

E200). PCNA assessment was evaluated by counting positive cells/total cells for each

follicle to obtain an index of positive cells. Between 34 and 427 granulosa cells per

follicle were evaluated.

2.8. Quantitative real-time PCR analysis for BAX, BCL2 in isolated secondary follicles

For RNA isolation, three pools of 10 isolated secondary follicles were collected

from each experimental group after 7 days of culture (Exp.1). The samples were stored

in microcentrifuge tubes (1.5 mL) with 100 µL Trizol at -80 ºC. Total RNA from follicles

were isolated and purified with Trizol® Plus Purification kit (Invitrogen, São Paulo,

Brazil). The RNA preparations were treated with DNase I and Pure Link RNA Mini Kit

(Invitrogen, São Paulo, Brazil). Complementary DNA (cDNA) was synthesized from the

isolated RNA using Superscript II RNase H-Reverse Transcriptase (Invitrogen, São

Paulo, Brazil). The qPCR reaction was performed in a final volume of 20 µL, containing

1 µL of each cDNA, 1 x Power SYBR Green PCR Master Mix (10 µL) (PE Applied

Biosystems, Foster City, CA, USA), 5.5 µL of ultrapure water, and 0.5 µM of both sense

and anti-sense primers. The gene-specific primers used for the amplification of different

transcripts are shown in Table 1. Transcript levels in follicular cells were normalized to

the content of peptidylprolyl Isomerase A (PPIA) and glyceraldehyde-3-phosphate-

dehydrogenase (GAPDH). Primer specificity and amplification efficiency were verified

for each gene. The qPCR cycling conditions consisted of an initial denaturation and

polymerase activation step at 94 ºC for 15 min, followed by 40 cycles of 15 s at 94 ºC, 30

s at 60 ºC, and 45 s at 72 ºC, and then a final extension for 10 min at 72 ºC. After

amplification, melting curve analysis was performed between 60 ºC and 95 ºC for all

genes. All amplifications were carried out in a Bio-Rad iQ5 (Hercules, CA, USA). The

96

delta-delta-CT method was used to transform threshold cycle values into normalized

relative expression levels (LIVAK; SCHMITTGEN, 2001).

2.9. In vitro maturation of caprine oocytes recovered from antral follicles

In the laboratory, approximately 399 cumulus oocytes complexes (COCs) from

70 ovaries were collected by slicing of ovaries. Oocytes with a compact cumulus mass

and a dark, evenly granulated cytoplasm were selected for IVM and washed three times

in base maturation medium (TCM-199+), consisting of TCM-199 supplemented with 1

µg/mL 17β- estradiol, 5 µg/mL LH, 0.5 µg/mL rFSH, 10 ng/mL EGF, 1 mg/mL BSA, 22

µg/mL pyruvate, 50 ng/mL IGF-I, and 100 µmol/L cysteamine, previously pre-

equilibrated at 39°C and 5% CO2 .in air. Groups of COCs were cultured in 500 µL of

designed media into each well of a 4-well multidish (Nunc, Roskilde, Denmark) for 24 h

at 39 °C, in 5% CO2.

2.10. Assessment of oocyte chromatin configuration and viability

Following maturation, the COCs were denuded mechanically and oocytes were

washed in PBS, then incubated in 100 µL droplets containing 4 µM calcein-AM, 2 µM

ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany), 0.5% of

glutaraldehyde and 10 µM Hoechst 33342 for 30 min.

The chromatin configuration was assessed by fluorescence microscopy (Nikon,

Eclipse 80i, Tokyo, Japan), and classified as abnormal chromatin configuration, when the

nucleus was pyknotic, compact or in a strange configuration; and normal chromatin

configuration was considered when the nucleus was in germinal vesicle (GV) or meiotic

resumption. Meiotic resumption was defined when the nucleus was in germinal vesicle

break down (GVBD), metaphase I (MI) or in metaphase II (MII) stages. Thereafter,

oocytes were also examined under a fluorescence microscope (Nikon, Eclipse 80i, Tokyo,

Japan) for evaluation of live/dead fluorescent staining. The emitted fluorescent signals of

calcein-AM and ethidium homodimer-1 were collected at 488 and 568 nm, respectively.

Oocytes were considered alive when the cytoplasm was stained positively with calcein-

AM (green) and chromatin was not labeled with ethidium homodimer-1 (red). Also,

oocytes were classified as viable when the cytoplasm was stained positively with calcein-

AM and they showed a normal chromatin configuration, as mentioned above. Moreover,

97

abnormal oocytes were divided in three categories: oocytes stained positively with

calcein-AM with abnormal chromatin were considered as alive with abnormal chromatin

configuration, or oocytes non stained with calcein-AM and stained with ethidium

homodimer were considered as non-alive; and finally non-alive plus alive with abnormal

chromatin configuration which formed the total number of abnormal oocytes.


For both experiments, data referring to continuous variables were initially

evaluated for homocedasticity and normal distribution of the residues by Bartlett’s and

Shapiro-Wilk tests, respectively. Confirmed both requirements underlying analysis of

variance, it was carried out considering a completely randomized design in a factorial

arrangement 5 x 2 (five treatments and two times of culture). When any main effect or

their interactions were significant, comparisons were further analyzed by Student-

Newman-Keuls test, being the results expressed as mean ± standard deviation. When

heterocedasticity was observed, even after transformation of data, non-parametric

Kruskal-Wallis test was applied. Data for discrete variables were analyzed by chi-square

test (or Fisher Exact Test when n<5) and results were expressed as percentages. In all

cases, a probability of P<0.05 indicated a significant difference.

3. Results and discussion

The present study utilized for the first time reproductive toxicity of in vitro

cultured isolated follicles and in vitro matured COC’s to assess the toxicity of A.

oncocalyx and its isolated compound onco A. In order to investigate the effects of

different drugs over folliculogenesis, there are various toxicological tests. In vitro cultures

have become a tool to analyze the effects of several components on the survival, growth

and maturation of follicles in many animal models (STEFANSDOTTIR et al., 2014). In

vitro experiments are of great importance as they provided appropriated information

about the effect of different compounds before in vivo testing. The in vitro culture of

caprine preantral follicles and the in vitro maturation of COCs showed to be appropriated

techniques to analyze the drugs effect over folliculogenesis.

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A total of 301 secondary follicles were analyzed before and after in vitro culture

in different treatments (Figure 1). The effects of the treatments on the percentage of

morphologically intact follicles and antrum formation are shown (Table 2). After 7 days

of culture there was a lower (P < 0.05) percentage of morphologically intact follicles and

antrum formation in DXR treatment compared to the other treatments (control, DMSO,

A. oncocalyx and onco A). Follicular diameter and growth rate of follicles after 7 days of

in vitro culture are shown (Table 3). There was a significant increase in follicular diameter

from D0 to D7 in all treatments except for onco A and DXR. In addition, we observed

that DXR significantly reduced the growth rate when compared to the other treatments

(control, DMSO, A. oncocalyx and onco A). The in vitro follicle culture system used in

the present study was effective to investigate the impact of the studied drugs (DXR, A.

oncocalyx and onco A) on in vitro folliculogenesis. The control medium used ensured the

maintenance of appropriate rates of follicle survival (92.54 %), growth (4.19 ± 6.12) and

antrum formation (59.70 %). These data are in agreement with previous studies reporting

the in vitro culture of caprine preantral follicles for 6 days, using the same culture medium

(ARAÚJO et al., 2011; DUARTE et al., 2013). In this study, A. oncocalyx and onco A

did not alter the folliculogenesis parameters such as survival, antrum formation and

growth rate. On the contrary, DXR caused a toxic effect on all these folliculogenesis end

points. A. oncocalyx and onco A were less harmful to in vitro secondary follicles than

DXR, although both drugs were able to maintain follicular morphology, without affecting

neither antrum formation, nor follicular growth. It is known that onco A and DXR both

have anti-carcinogenic effects (PESSOA et al., 2004). The fraction of A. oncocalyx and

onco A in different lineages of cells, especially in tumor cell lines, had a similar effect

when compared to DXR (PESSOA et al., 2000, 2003, 2004). DXR and onco A have been

shown to exhibit cytotoxicity in the G, G1/S and S phases of cell division, however, only

DXR exhibited genotoxicity in these cells (PESSOA et al., 2003). This genotoxicity,

caused by DXR, could explain the decrease in the percentage of morphologically intact

follicles and antrum formation found in this study.

The percentage of TUNEL positive follicles (TPF) was higher (P < 0.05) for the

DXR treatment when compared to non-cultured control. There was no difference (P >

0.05) in the percentage of TPF among the other treatments. The number of PCNA positive

granulosa cells were lower (P <0.05) for the DXR treatment when compared to the other

treatments (control, DMSO, A. oncocalyx and onco A) (Table 4). Additionally, DXR was

99

the only treatment to cause a decrease (P < 0.05) in the relative BAX:BCL2 mRNA ratio

(Figure 2). More studies are needed to discern the mechanism of action of both drugs.

Recent studies revealed that DXR induced ovarian toxicity, which was observed by a

reduction in ovulation rate and the size of the ovary (OKTEM; OKTAY, 2007; BAR-

JOSEPH et al., 2010; BEN-AHARON et al., 2010). DXR elicits apoptosis by various

mechanisms in a variety of cells. It can be accumulated in both the nucleus and

mitochondria, and induces chromosomal destruction by inhibiting topoisomerase-II

(TOKARSKA-SCHLATTNER et al., 2006). DXR can also interfere with mitochondrial

function and initiate an intrinsic pathway of apoptosis by reducing the mitochondrial

membrane potential and releasing cytochrome C (BAR-JOSEPH et al., 2010).

In this study, there was a decrease (P < 0.05) in the percentage of viable oocytes

when they were treated with DXR, A. oncocalyx and onco A. The percentage of non-alive

oocytes was significantly higher (P < 0.05) in the onco A treatment than DXR and A.

oncocalyx treatments. The opposite was observed for the percentage of alive oocytes with

abnormal chromatin configuration. Compared to its vehicle (DMSO), onco A and DXR

treatments reduced (P < 0.05) the percentage of oocyte meiotic resumption, but they were

similar (P > 0.05) to the control treatment. In addition, the MII rates were similar (P >

0.05) among the treatments (Table 05). The in vitro maturation culture system used in the

present study was adequate to investigate the impact of the studied drugs (DXR, A.

oncocalyx and onco A) on in vitro maturation. The control medium used ensured the

maintenance of appropriate rates of viability (90.67 %) and meiotic resumption (92.65

%). These data are in agreement with previous studies made by our group, reporting the

in vitro maturation of caprine COCs, using the same culture medium. In this study, DXR,

A. oncocalyx and onco A negatively affected the oocyte viability (Figure 1). It is known

that DXR can induce cellular apoptosis. Moreover, a study evaluating different

concentrations (1 to 100 µg/mL) of a quinone fraction of A. oncocalyx (containing 80%

of onco A) in sea urchin eggs reported that the cleavage of eggs was inhibited in a

concentration-dependent manner, and when a concentration of 30 µg/mL was used it

caused a total destruction (100%) of embryos (COSTA-LOTUFO et al., 2002).

Only DXR and onco A caused a harmful effect on meiotic resumption. This

finding may be due to the higher sensitivity of the COCs to a toxic compound. The

presence of alive oocytes with abnormal chromatin configuration implies the occurrence

of apoptosis (BAR-JOSEPH et al., 2010). Moreover, only DXR and A. oncocalyx

100

exhibited an increase in oocytes with abnormal chromatin configuration, showing a less

harmful effect of onco A. This result may be due to the impurity of the fraction of A.

oncocalyx which contains 80% of onco A (FERREIRA et al., 2004), and 20% of other

substances that may be more toxic than onco A alone. Also, Pessoa et al. (PESSOA et al.,

2003), showed that even though onco A and DXR have cytotoxicity in lymphocytes, only

DXR present genotoxicity, proving to be more toxic. It is important to highlight that the

DMSO treatment (i.e., the vehicle used for the dilution of DXR, A. oncocalyx and onco

A) was similar to the α-MEM+ cultured control. This result shows that DMSO by itself

was not responsible for the negative effect of the tested drugs in any of the studied end

points.

4. Conclusions

In conclusion, under our culture conditions, A. oncocalyx and onco A do not have

a toxic effect on isolated secondary follicles and maturation rates on in vitro matured

COCs, however, these drugs affect the oocyte viability after in vitro maturation. In

addition, the less harmful effect of onco A than DXR on caprine secondary follicle

survival and oocytes with normal chromatin configuration may encourage future studies

involving the use of this drug for cancer treatment in women.

5. Acknowledgments


authors thank Francisco Leo Aguiar, Victor Macedo Paes, Denise Damasceno Guerreiro,

Renato Félix da Silva and Naiza Arcângela Ribeiro de Sá for assistance with this study.

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103

7. Figures and tables

Table 1. Oligonucleotide primers used for PCR analysis of goat secondary follicles

Target gene Primer sequence (5'→3')

Sense (S) Genbank

accession nos. Antisense (AS)

GAPDH ATGCCTCCTGCACCACCA S GI: 298676424

(Ovis aries) AGTCCCTCCACGATGCCAA AS

PPIA TCATTTGCACTGCCAAGACTG S GI:548463626

(Capra hircus) TCATGCCCTCTTTCACTTTGC AS

BAX TTTTGCTTCAGGGTTTCATCCAGGA S GI:926714830

(Capra hircus) CAGCTGCGATCATCCTCTGCAG AS

BCL2 GTTTTCCGACGGCAACTTC S

GI:354549710

(Capra hircus)

104

Figure 1. Isolated secondary follicles before (a) and after 7 days of culture in α-MEM+

alone (cultured-control) (b) or supplemented with DMSO (c), DXR (d), A. oncocalyx (e)

and onco A (f). Oocytes after in vitro maturation in TCM199+ (control) (g) or

supplemented with DMSO (h), DXR (i), A. oncocalyx (j) or onco A (k).

Figure 2. Relative mean (± SEM) of BAX:BCL2 mRNA ratio in cultured isolated

secondary follicles for 7 days in α-MEM+ alone (cultured-control) or supplemented with

DMSO, DXR, A. oncocalyx and onco A. Different letters denote significant differences

(P < 0.05).

105

Table 2. Percentage of morphologically intact secondary follicles, and antrum formation

after in vitro culture for 7 days in α-MEM+ (control) or supplemented with DMSO, DXR,

A. oncocalyx or onco A.

Treatments (n) % Morphologically intact follicles % Antrum formation

Control (n = 67) 92.54 (62/67) A 59.70 (40/67) A

DMSO (n = 58) 89.66 (52/58) A 50.00 (29/58) A

DXR (n = 59) 55.93 (33/59) B 16.95 (10/59) B

A. oncocalyx (n = 59) 86.44 (51/59) A 57.63 (34/59) A

Onco A (n = 58) 84.48 (49/58) A 55.17 (32/58) A

A,B Distinct capital letters represent significant differences among experimental treatments (P <

0.05). n Total number of analyzed follicles per treatment

Table 3. Follicular diameter (on day 0 and 7) and growth rate (mean ± SEM) of isolated

secondary follicles after in vitro culture in α-MEM+ (control) or supplemented with

DMSO, DXR, A. oncocalyx or onco A

Treatments D0 D7 Growth rate

Control 156.87 ± 39.13 Ab 184.41 ± 53.16 Aa 4.19 ± 6.12 A

DMSO 155.26 ± 34.43 Ab 177.99 ± 53.41 Aa 2.96 ± 6.87 A

DXR 151.59 ± 38.55 Aa 148.12 ± 39.36 Ba -0.49 ± 3.80 B

A. oncocalyx 161.44 ± 38.24 Ab 188.87 ± 52.56 Aa 4.03 ± 6.62 A

Onco A 165.20 ± 44.77 Aa 178.05 ± 51.22 Aa 1.85 ± 6.79 A

A,B Distinct capital letters represent significant differences among treatments within the same day

of culture. a,b Different lowercase letters represent significant differences between days of culture

within the same treatment. (P < 0.05).

106

Table 4. PCNA test and TUNEL assay of non-cultured or in vitro cultured isolated

secondary follicles for 7 days in α-MEM+ (control) or supplemented with DMSO, DXR,

A. oncocalyx or onco A.

Treatments PCNA TUNEL

Non-cultured control 87.45 ± 7.32 A 16.67 % (1/6) B

Control 89.72 ± 8.83 A 33.33 % (2/6) AB

DMSO 94.20 ± 6.00 A 33.33 % (2/6) AB

DXR 16.54 ± 9.70 B 100.00 % (4/4) A

A. oncocalyx 93.20 ± 4.51 A 60.00 % (3/5) AB

Onco A 94.07 ± 6.71 A 50.00 % (3/6) AB

Distinct capital letters represent significant differences among experimental treatments (P < 0.05).

107

Table 5. Viable and non-viable oocytes rates, germinal vesicle (GV), meiotic resumption and metaphase II (MII) rates, after in vitro

maturation in TCM199+ (control) or supplemented with DMSO, DXR, A. oncocalyx or onco A of COCs recovered from antral follicles.

NON-VIABLE OOCYTES

VIABLE

OOCYTES

MATURATION RATES OF VIABLE OOCYTES

Treatments TOTAL Non-alive Alive # TOTAL GV

Meiotic

GVBD MI MII Resumption*

(n) % % % % % % % % %

Control 75 57.14 AB 42.86 AB 9.33 B 90.67 A 7.35 AB 92.65 AB 5.88 A 29.41 A 57.35 A

(4/7) (3/7) (7/75) (68/75) (5/68) (63/68) (4/68) (20/68) (39/68)

DMSO 84 69.23 AB 30.77 AB 15.48 B 84.52 A 4.23 B 95.77 A 8.45 A 30.99 A 56.34 A

(9/13) (4/13) (13/84) (71/84) (3/71) (68/71) (6/71) (22/71) (40/71)

DXR 77 46.43 B 53.57 A 36.36 A 63.64 B 14.29 A 85.71 B 2.04 A 36.73 A 46.94 A

(13/28) (15/28) (28/77) (49/77) (7/49) (42/49) (1/49) (18/49) (23/49)

A. oncocalyx 77 44.12 B 55.88 A 44.16 A 55.84 B 13.95 AB 86.05 AB 2.33 A 34.88 A 48.84 A

(15/34) (19/34) (34/77) (43/77) (6/43) (37/43) (1/43) (15/43) (21/43)

Onco A 86 71.79 A 28.21 B 45.35 A 54.65 B 17.02 A 82.98 B 4.26 A 31.91 A 46.81 A

(28/39) (11/39) (39/86) (47/86) (8/47) (39/47) (2/47) (15/47) (22/47)

A,B Distinct capital letters represent significant differences among treatments (P < 0.05)

n Total number of analyzed oocytes per treatment

* Includes GVBD, MI and MII oocytes

# Oocytes stained positively with calcein-AM (green) that presented an abnormal chromatin configuration (marked with Hoechst).

108

9 AUXEMMA ONCOCALYX E SEU COMPOSTO ATIVO ONCOCALYXONA A

PREJUDICAM A COMPETÊNCIA DE DESENVOLVIMENTO OOCITÁRIO

IN VITRO EM SUÍNOS, MAS SÃO MENOS PREJUDICIAIS DO QUE A

DOXORRUBICINA

“Auxemma oncocalyx and its active compound oncocalyxone A impair in vitro porcine

oocyte developmental competence but are less detrimental than Doxorubicin.”

Periódico: Planta medica (submetido) (ISSN: 0032-0943)

Qualis A2

109

RESUMO

Diversas drogas anticancerígenas, como a doxorrubicina (DXR), possuem menor

especificidade de ação, resultando em efeitos indesejáveis. Plantas são uma excelente

fonte de substâncias na pesquisa de novas drogas. Auxemma oncocalyx (A. oncocalyx) e

o seu componente, a oncocalixona A (Onco A) possuem atividade antitumoral e são

menos tóxicos em relação à DXR no que se refere aos parâmetros reprodutivos.

Entretanto, não existem estudos a respeito da ação dessas drogas no âmbito da

competência oocitária in vitro e desenvolvimento embrionário no modelo suíno. Com

isso, o objetivo deste estudo consistiu em avaliar os efeitos da adição de A. oncocalyx e

Onco A durante a maturação in vitro (MIV) de oócitos (Experimento 1) ou cultivo in

vitro de embriões (Experimento 2) no modelo suíno. Para o experimento 1, complexos-

cumulus-oócitos (CCOs) foram distribuídos no meio de MIV de modo isolado (controle)

ou suplementado com DXR (0,3 ug/mL), A. oncocalyx (1,2 ug/mL) e Onco A (1 ug/mL).

Em seguida, oócitos foram submetidos à fertilização in vitro (FIV) e posterior cultivo in

vitro de embriões. Para o segundo experimento, CCOs foram submetidos à MIV e FIV,

onde seus presumíveis zigotos foram cultivados com DXR, A. oncocalyx ou Onco A por

7 dias. A Viabilidade, maturação, fertilização e desenvolvimento embrionário foram

parâmetros avaliados em ambos os experimentos. No experimento 1, DXR, A.

oncocalyx e Onco A reduziram de modo significativo (P<0,05) a viabilidade oocitária e

eficácia da MIV. Onco A aumentou de modo significativo (P<0,05) a retomada da

meiose. Entretanto, os oócitos ficaram bloqueados no estágio de MII. Após a FIV, todas

as drogas reduziram de modo significativo (P<0,05) a viabilidade, eficiência da FIV e

percentual de zigotos clivados. No entanto, apenas DXR reduziu a percentagem de

blastocistos. No experimento 2, todas as drogas reduziram de modo significativo (P<0,05)

a percentagem de penetração, mas apenas DXR e Onco A reduziram (P<0,05) a eficiência

da FIV. DXR e A. oncocalyx reduziram de modo significativo (P<0,05) o percentual de

zigotos clivados, mas não afetaram a formação de blastocistos. Em conclusão, a adição

de DXR durante a MIV ou CIV afetam negativamente a eficácia da FIV e taxa de

clivagem. Em adição, a exposição dos CCOs à DXR somente durante a MIV foi mais

prejudicial à viabilidade oociátia e formação de blastocisto quando comparado à A.

oncocalyx e Onco A.

Palavras-chave: Auxemma oncocalyx. Oncocalyxone A. Doxorrubicina. Maduração in

vitro. Fertilização in vitro. Desenvolvimento embrionário.

110

Auxemma oncocalyx and its active compound oncocalyxone A impair in vitro porcine

oocyte developmental competence but are less detrimental than Doxorubicin

Leiva-Revilla J. 1, Maside C. 1, Vieira L. 1, Cadenas J.1, Ferreira A.A. 1, Paes V.M. 1, Agiar

F.L. 1, Celestino J.J.H. 2, Alves B. 1, Pessoa O.D.L. 3, Apgar G.A. 4, Toniolli R. 5,

Rodrigues A.P.R. 1, Figueiredo J.R. 1*.

1 Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA).

Universidade Estadual do Ceará, Fortaleza-CE, Brazil.

2 Institute of Health Sciences. Universidade da Integração Internacional da

Lusofonia Afro-Brasileira. Acarape-CE, Brazil.

3 Department of Organic and Inorganic Chemistry. Universidade Federal do Ceará, Centro

de Ciências. Fortaleza-CE, Brazil

4 Professor of Department of Animal Science, Food and Nutrition, Southern Illinois

University-Carbondale, USA

5 Laboratory of Swine Reproduction and Semen Technology. Universidade Estadual do

Ceará, Fortaleza-CE, Brazil.

* Corresponding address:

Laboratório de Manipulação de Oócitos e Folículos Pré-Antrais (LAMOFOPA).

Universidade Estadual do Ceará (UECE). Av. Silas Munguba, 1700, Campus do Itaperi.

Fortaleza – CE – Brasil. CEP: 60740 903

Tel.: +55.85. 3101.9852; Fax: +55.85.3101.9840

E-mail address: [email protected]

111

ABSTRACT

Most anticancer drugs like doxorubicin (DXR) have low specificity that results in

undesirable effects. Plants are excellent sources when searching for new drugs. Auxemma

oncocalyx (A. oncocalyx) and its main component oncocalyxone A (onco A) have anti-

tumoral activity and are less toxic than DXR in reproductive parameters. However, there

are no studies on the action of these drugs regarding the porcine in vitro oocyte

competence and embryo development. The aim of this study was to evaluate the effect of

A. oncocalyx and onco A exposure during in vitro maturation (IVM) of oocytes

(Experiment 1) or in vitro embryo culture (IVC) (Experiment 2) on the oocyte

developmental competence. For experiment 1, COCs were distributed in IVM medium

alone (control) or supplemented with DXR (0.3 g/mL), A. oncocalyx (1.2 g/mL) and

onco A (1 g/mL). Then, oocytes were submitted to in vitro fertilization (IVF) and in

vitro embryo culture. For experiment 2, COCs were submitted to IVM and IVF and the

presumptive zygotes were cultured with DXR, A. oncocalyx and onco A for 7 days.

Viability, maturation, fertilization and embryo developmental parameters were evaluated

in both experiments. In experiment 1; DXR, A. oncocalyx and onco A reduced (P<0.05)

oocyte viability and IVM efficiency. Onco A increased (P<0.05) the meiotic resumption,

however, oocytes were arrested at MI. After IVF, all the drugs reduced (P<0.05) viability,

IVF efficiency and percentage of cleaved embryos, nevertheless, only DXR decreased

the percentage of blastocyst. In experiment 2; all drugs reduced (P<0.05) the percentage

of penetration, but only DXR and onco A decreased (P<0.05) IVF efficiency. DXR and

A. oncocalyx decreased (P<0.05) the percentage of cleaved embryo, but had no effect on

blastocyst formation. In conclusion, the addition of DXR during IVM or IVC negatively

affected the IVF efficiency and cleavage rate. In addition, the exposure of COCs to DXR

only during IVM was more detrimental to oocyte viability and blastocyst formation than

A. oncocalyx and onco A.

Keywords: Auxemma oncocalyx; Oncocalyxone A; Doxorubicin; in vitro maturation; in

vitro fertilization; embryo development

112

Abbreviations

Auxemma oncocalyx A. oncocalyx

cGMP-dependent protein kinase PKG

Cumulus oocyte complexes COCs

Cyclic guanosine monophosphate cGMP

Double-strand breaks DSBs

Doxorubicin DXR

Germinal vesicle GV

Germinal vesicle break down GVBD

Hours post insemination hpi

In vitro embryo culture IVC

In vitro fertilization IVF

In vitro maturation IVM

Metaphase 1 MI

Metaphase 2 MII

Oncocalyxone A Onco A

Poly (AND-ribose) polymerase PARP

Topoisomerase II TOPO II

Topoisomerases TOPOs

113

1. Introduction

Cancer is a target of research for the development or discovery of new forms of

treatments [1]. Many drugs used in cancer chemotherapy, like doxorubicin (DXR), a

widely used drug for different cancer types[2], have low specificity that results in

undesirable effects. Studies show that DXR cause toxicity in both primordial follicles and

growing ovarian follicles, triggering follicular and oocyte apoptosis [3], and eventually

affect human fertility. Therefore, plants are excellent sources of raw material when

searching for new anticancer drugs [4].

AuxemmA. oncocalyx (A. oncocalyx) is a common tree found in the state of Ceará

in Northeast Brazil [5]. It has been widely used in folk medicine as an adjunctive

treatment of injuries such as wounds and cuts [5,6]. Some studies have suggested that this

plant has biological activities such as analgesic, antioxidant, antitumor and anti-

inflammatory effects [6–9].Oncocalyxone A (onco A) is A. oncocalyx’s active compound.

Onco A has high antioxidant activity [6] and an anti-proliferative effect on tumor cell

cultures [10]. Studies have suggested onco A as a possible anticancer compound since it

presents antitumor and cytotoxic activity in human leukemia cells, and other cell cancer

lines, without causing genotoxicity [11] as most anticancer drugs.

Little is known about reproductive toxicity of A. oncocalyx and onco A in

mammals. In recent pioneer studies conducted by our group with caprine preantral

follicles cultured in vitro enclosed in ovarian cortical tissue, A. oncocalyx and onco A

affected in vitro caprine early folliculogenesis in a concentration-dependent manner [12].

However, no toxic effect of A. oncocalyx and onco A was observed on in vitro

development of late caprine isolated secondary follicles. In contrast, these drugs affected

the cumulus-oocyte complexes (COCs) viability after in vitro maturation but not the

metaphase II rates (Leiva-Revilla et al., 2016 b – under review). In both studies, DXR

was used as positive (toxic) control and presented a more toxic effect than A. oncocalyx

and onco A. These results suggest that A. oncocalyx and onco A despite of having

anticancer effects they are, apparently, less harmful to reproductive parameters than

commercial drugs, such as DXR.

Normal embryonic development is preceded by a sequence of coordinate events

during maturation and fertilization. The mechanism of oocyte maturation encompasses

interactions between the oocyte and its surrounding cumulus cells, which synchronizes

114

meiosis with structural and molecular changes in the ooplasm, enabling the oocyte to

support proper fertilization and subsequent embryo development [13]. Consequently, it

is of high significance to study the toxic effect of new drugs over in vitro maturation,

fertilization and embryo development.

To the best our knowledge, there is no information about the influence of A.

oncocalyx and onco A on the in vitro embryo development in mammals, including pigs.

Compared to the other species, the porcine seems to be a suitable animal model for

humans, due to the ovarian similarities [14].In addition, the advantage of using pig ovaries

is that the ovaries are from animals at similar age, breed and controlled nutrition. Thus,

the porcine specie has been quite used as a model for human oocytes in toxicity tests [14].

Therefore, the aim of this study was to evaluate the effect of A. oncocalyx and onco A

exposure during in vitro maturation of oocytes (Experiment 1) or in vitro embryo culture

(Experiment 2) on the oocyte developmental competence, investigating the following end

points: oocyte viability, maturation rates and efficiency, in vitro fertilization parameters

and percentage of cleaved embryos and blastocyst formation.

2. Results

2.1. Experiment 1. Effect of A. oncocalyx and onco A on IVM of porcine COCs and

subsequent embryo development.

The rates of oocyte viability and maturation after exposure to DXR, A. oncocalyx

and onco A are shown (Table 01). Take into account the total number of COCs submitted

to IVM, the DXR, A. oncocalyx and onco A treatments showed a significant lower

percentage of oocyte viability and maturation than control. Except for oocyte viability, A.

oncocalyx and onco A showed similar results for the aforementioned endpoints being

both higher (P < 0.05) than DXR. However, when only viable oocytes were considered

to calculate the maturation rate no differences were observed among treated groups

(DXR, A. oncocalyx and onco A). To determine the meiotic resumption and MI rates only

viable oocytes were used. The percentage of meiotic resumption in the onco A treatment

and rate of MI in the A. oncocalyx and onco A treatments were similar to DXR but higher

(P < 0.05) than control.

115

The exposure of COCs to DXR, A. oncocaly and onco A during IVM reduced

significantly the IVF efficiency when compared to the control treatment (Table 2).

However, when only matured oocytes were taken into account the penetration and

monospermy rates as well as the number of spermatozoa/oocyte were similar (P > 0.05)

among the treatments.

When the COCs were exposed to the three tested drugs during IVM (Figure 2),

DXR, A. oncocalyx and onco A treatments showed lower (P < 0.05) cleaved rates

compared to control. This endpoint was higher in the onco A treatment than DXR and A.

oncocalyx treatments. With regard to blastocyst rate, lower (P < 0.05) values were

observed in the DXR treatment.

2.2. Experiment 2. Effect of A. oncocalyx and onco A on the in vitro culture of porcine

embryos.

Contrary to the experiment 1, where the COCs were exposed to the tested drugs

during IVM, in the experiment 2, only the in vitro presumptive zygotes were exposed to

DXR, A. oncocalyx and onco A for 18 hpi (Table 3). There was no difference (P > 0.05)

between control group and other treatments regarding to viability and maturation rates.

However, DXR and onco A showed a lower percentage (P <0.05) of viability than A.

oncocalyx. All the tested treatments reduced (P < 0.05) the penetration rate compared to

control treatment. Onco A, but not A. oncocalyx reduced significantly the monospermy

and IVF efficiency, and increased (P < 0.05) the number of spermatozoa/oocyte. The IVF

efficiency was also reduced (P < 0.05) when DXR was added to the control medium.

The exposure of presumptive zygotes to onco A did not change either the cleavage

or blastocyst rates comparing control (Figure 3). The addition of DXR and A. oncocalyx

to the control medium only reduced (P < 0.05) the cleavage rate.

3. Discussion and conclusion

To our knowledge, the present study demonstrated for the first time the effect of

A. oncocalyx and its isolated compound, onco A, on the in vitro maturation of porcine

oocytes and subsequent in vitro embryo development.

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When the three tested drugs exposure occurred only during IVM (Experiment 1),

DXR, A. oncocalyx and onco A had a detrimental effect on the oocyte viability, being

DXR the most toxic with only 12.83 % viable oocytes after 44h of exposure. It is known

that DXR acts on several levels by different molecular mechanisms including an

interaction with iron, upsetting calcium homeostasis, altering the activity of intracellular

or intra-mitochondrial oxidant enzymes, and binding to topoisomerases (TOPOs)

promoting their dysfunction leading to DNA damage and apoptosis [17]. Specifically,

DXR acts by inhibiting Topoisomerase II (Topo II). In the female, Topo II is required for

chromosome separation during oocyte meiotic maturation, but is dispensable for

resumption of meiosis [18]. On the other hand, A. oncocalyx and onco A also disrupt

oocyte viability and caused damage in the chromatin configuration. Sbardelotto [19]

showed that, in human promyelocytic leukemia line (HL-60) cells, onco A activates first

the intrinsic apoptotic pathway by caspase 8, and then the extrinsic pathway by caspase 3

and 7. Contrary to DXR, onco A does not affect the TOPOs. However, in HL-60 cells,

onco A cleaved poly (ADP-ribose) polymerase (PARP) [19]. PARP binds and repair

DNA-strand breaks generated by genotoxic agents. Likewise, PARP is implicated in the

regulation of a wide range of important cellular processes including transcriptional

regulation, chromatin modification, cellular homeostasis, and cell proliferation and death

[20]. Therefore, cleaved PARP results in an oocyte proapoptotic protein [21]. A study

conducted in porcine showed that the cleavage of PARP1 was strongly implicated in

follicular development and atresia of fetal, neonatal, and adult porcine ovaries [20]. In

addition, PARP-1 synthesize PAR, which is required for assembly and function of the

bipolar spindle [22]. PARP-1 also mediates the regulation of centrosome duplication and

chromosomal stability. The inhibition of PARP-1 is associated with mislocalization of

centromeric and centrosomal proteins, defective chromatin modifications and genomic

instability characterized by loss of mitotic checkpoint integrity [23].

In the present study, the IVM efficiency was compromised in all treatments

compared to control treatment. Interestingly, onco A increased the meiotic resumption

rates. However, these oocytes were arrested at the MI stage. Anticancer drugs can cause

double-strand breaks (DSBs). These DSBs do not arrest mouse oocytes in the

G2/prophase but, instead, allow them to progress to the MI stage [24]. The exposure of

oocytes to genotoxic agents, such as PARP inhibitors, causes failures in the spindle

assembly checkpoint, leading to an oocyte meiotic arrest at MI [24]. Another study

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showed that damage in the microtubules, main structural elements of the spindle, result

in oocytes arrest at the MI stage during in vitro maturation in mouse [25]. Our results

suggest that onco A might be affecting the oocyte chromatin configuration, through

PARP inhibition, leading to failures in the spindle assembly checkpoint and therefore

causing this meiotic arrest at the MI stage.

In the present study, the exposure of COCs to the tested drugs only during IVM

negatively affected the IVF efficiency. Even tough DXR, A. oncocalyx and onco A

reduced embryo cleavage rate, only DXR showed a more toxic effect on blastocyst

development. DXR elicits apoptosis by various mechanisms in a variety of cells. DXR is

capable to accumulate in both nucleus and mitochondria and induce chromosomal

obliteration by inhibiting Topo-II. In oocytes, it can interfere with mitochondrial function

and start the intrinsic pathway of apoptosis via the mitochondria by reducing the

mitochondrial membrane potential and releasing cytochrome C [26]. Impaired

mitochondrial function lead to improper fertilization and a reduction of embryo

development [27].

When DXR, A. oncocalyx and onco A were added only during the in vitro embryo

culture (Experiment 2), all drugs negatively affected the penetration rate evaluated after

18 hpi. However, DXR and onco A showed a detrimental effect on IVF efficiency.

Nonetheless, only onco A augmented the percentage of spermatozoa per oocyte. Ferreira

et al. [28] showed that onco A was able to inhibit platelet aggregation by increasing the

cyclic guanosine monophosphate (cGMP) levels in platelets by a synergistic mechanism,

combining increased production and reduced degradation of cGMP [28]. In porcine,

cGMP activates cGMP-dependent protein kinase (PKG). This pathway plays an essential

role in acrosome reaction, which enables the spermatozoa to penetrate the zona pellucida,

and therefore, to fuse with the oocyte plasma membrane [29]. Zhang [30] observed that

when a cGMP analog, atrial natriuretic peptide (ANP), was added during IVF of frozen-

thawed giant panda sperm with porcine salt-stored oocytes, it resulted in a higher

proportion of oocytes with spermatozoa in the zona pellucida and perivitelline space, and

a higher average number of spermatozoa/oocyte.

In experiment 2, DXR, A. oncocalyx and onco A negatively affected the cleavage

rate. Wang et al [31] showed that DXR blocked pre-implantation development in early

mouse embryos by altering apoptosis-related gene expression, Bcl2l1 and Casp3, and

118

inactivating DNA repair by PARP. In the same study, the authors found out that DXR

arrested zygotes at the 1-cell stage by disruption of DNA and of the cytoskeleton. In

addition, it is known that during blastocyst formation, the inhibition PARP suppresses

selective autophagic degradation of ubiquitinated proteins, which contributes to apoptosis

[32]. Thus, the interaction between PARP and autophagy influences the quality of in vitro

produced embryos in porcine [33]. Due to the fact that onco A affects PARP in HL-60

cells [19], and that A. oncocalyx contains 80% of onco A in its composition [9], both

drugs could be affecting embryonic competition by this pathway. Moreover, a study

evaluating different concentrations (1 to 100 µg/mL) of a quinone fraction of A. oncocalyx

in sea urchin eggs reported that the cleavage of eggs was inhibited in a concentration-

dependent manner [34]. Despite the fact that DXR, A. oncocalyx and onco A reduced

porcine embryo cleavage rate, they did not affect blastocyst development, showing that

porcine blastocyst tend to be more resistant to toxic agents [35].

In conclusion, the addition of DXR during IVM or IVC negatively affected the

IVF efficiency and cleavage rate. In addition, the exposure of COCs to DXR only during

IVM was more detrimental to oocyte viability and blastocyst formation than A. oncocalyx

and onco A.

4. Materials and methods

4.1. Culture media

All chemicals used in the present study were purchased from Sigma Chemical Co.

(St. Louis, MO, USA) unless otherwise indicated. The medium used for the collection of

cumulus-oocyte complexes (COCs) and for washing was Dulbecco’s phosphate-buffered

saline (DPBS) medium composed of 136.89 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4

and 1.46 mM CaCl2·2H2O supplemented with 4 mg/mL bovine serum albumin (BSA),

0.34 mM sodium pyruvate, 5.4 mM D-glucose and 70 µg/mL kanamycin (mDPBS). The

oocyte maturation medium was modified-TCM 199supplemented with 150 µM

cisteamine and 10 ng/m Lepidermal growth factor(TCM-199+) [15]. The basic medium

used for fertilization was essentially the same as that used by Abeydeera and Day [16].

This medium, designated as a modified Tris-buffered medium, consisted of 113.1 mM

NaCl, 3 mM KCl, 7.5 mM CaCl2·2H2O, 20 mM Tris (crystallized free base), 11 mM

119

glucose and 5 mM sodium pyruvate supplemented with 2 mM caffeine and 0.2% BSA.

The embryo culture medium was a sequential medium based on NCSU-23 supplemented

with 0.4% BSA[15].

4.2. Isolation of onco A from A. oncocalyx

The obtaining of the A. oncocalyx and onco A has previously been described by

Pessoa et al.[11]. Briefly, A. oncocalyx was collected and identified by Dr. Maria Iracema

B. Loiola of the Department of Biology of Federal University of Ceará. Onco A

(C17H18O5) was extracted from woody parts of A. oncocalyx (Boraginaceae) by

phytochemical extraction methods using organic solvents, and was isolated and purified

by crystallization and recrystallization. It is noteworthy that the fraction of A. oncocalyx

contains 80 % of onco A [9], therefore the concentration of A. oncocalyx was in equal

proportion of onco A. A. oncocalyx and onco A were diluted with DMSO as a vehicle.

The concentrations of A. oncocalyx and onco A were chosen based on previous studies

performed in our laboratory [12].


Experiment 1: Effect of A. oncocalyx and onco A on IVM of porcine COCs and

subsequent embryo development

In this experiment, the effect of DXR, A. oncocalyx and onco A on in vitro

maturation (IVM) of porcine COCs and subsequent embryo development was evaluated

(Figure 1). Immediately after oocyte recovery, COCs were allocated into four treatments

to carry out IVM: I) TCM-199+ alone (control), or supplemented with II) 0.3 g/mL

DXR; III) 1.2 g/mL A. oncocalyx or IV) 1 g/mL onco A. After IVM, oocyte chromatin

configuration and viability were assessed. Moreover, oocytes from each group were

pooled, exposed to spermatozoa and cultured for 18 hours post insemination (hpi) to

assess fertilization parameters or for 7 (168 hpi) days to evaluate embryo development.

Experiment 2: Effect of A. oncocalyx and onco A on the in vitro culture of porcine

embryos.

COCs were submitted to IVM and IVF as described for experiment 1 (control

group), and presumptive zygotes were randomly allocated into four treatments for in vitro

embryo culture (IVC): I) NCSU-23 alone (control) or supplemented with II) 0.3 g/mL

120

DXR; III) 1.2 g/mL A. oncocalyx or IV) 1 g/mL onco A. The presumptive zygotes

were cultured for 18 hpi to assess fertilization parameters or for 7 days (168 hpi) to

evaluate embryo development (Figure 1).

4.4. Oocyte collection and IVM

Ovaries were obtained from prepuberal gilts at a local slaughterhouse and were

transported in 0.9% NaCl containing 70 µg/mL kanamycin, at 33°C within 1 h. In the

laboratory, COCs were aspirated from medium-sized follicles (3 to 6 mm in diameter)

using an 18-gauge needle connected to a 10-mL disposable syringe. Oocytes with a

compact cumulus mass and a dark, evenly granulated cytoplasm were washed three times

in maturation medium, and 50–60 oocytes were transferred into each well of a 4-well

multidish (Nunc, Roskilde, Denmark) containing 500-µL of maturation medium

supplemented with 10 IU/mL pregnant mare's serum gonadotropin and 10 IU/mL human

chorionic gonadotropin for 20–22 h. The oocytes were then incubated for another 20–22

h in maturation medium without hormones. Oocyte maturation was carried out under

mineral oil at 39ºC in a humidified atmosphere of 5% CO2 in air. After maturation, COCs

were mechanically denuded in maturationmedium and washed twice in maturation

medium.

4.5. In vitro fertilization and embryo culture.

Denuded oocytes were washed three times in pre-equilibrated fertilization

medium and fertilized as previously described [15].Briefly, groups of 30 denuded oocytes

were then placed in 50-µL drops of fertilization medium in a 35x10-mm Petri dish under

mineral oil and held at 38.5ºC in an atmosphere of 5% CO2 in air for approximately 30

min until the addition of spermatozoa. Pool of freshly ejaculated semen diluted in

extender from a three boars was obtained from a local breeding station. The semen was

washed three times by centrifugation at 1900 x g for 3 min in mDPBS. The resulting

pellet was re-suspended in fertilization medium, and after the appropriate dilution, 50 µL

of this sperm suspension was added to a 50µL drop of fertilization medium containing

the oocytes. The spermatozoa:oocyte ratio was 2000:1. The gametes were co-incubated

at 38.5ºC in a humidified atmosphere of 5% CO2 in air for approximately 4 h.

Presumptive zygotes were removed from the fertilization medium and washed

three times in pre-equilibrated embryo culture medium. The zygotes were then transferred

121

to a 4-well multidish (30 zygotes per well), with each well containing 500 µL of the same

medium under mineral oil, and were cultured at 38.5ºC in a humidified atmosphere of 5%

CO2 in air. Presumptive zygotes were cultured for the first 2 days (Day 0 = day of

fertilization) in glucose-free NCSU-23 supplemented with 0.33 mM pyruvate and 4.5 mM

lactate and then in fresh NCSU-23 medium containing 5.5 mM glucose until day 7.

4.6. Assessment of oocyte chromatin configuration, viability, sperm penetration and

embryo development

To evaluate maturation and fertilization parameters, denuded oocytes and

presumptive zygotes were washed in PBS-BSA, and incubated in 500 µL droplets

containing 4 µM calcein-AM, 2 µM ethidium homodimer-1 (Molecular Probes,

Invitrogen, Karlsruhe, Germany), 0.5% of glutaraldehyde and 10 µM Hoechst 33342 for

15 min.

The maturation rate was assessed at 44h of IVM. The chromatin configuration

patterns were the following: abnormal chromatin configuration, germinal vesicle (GV) or

meiotic resumption. Meiotic resumption was defined when the nucleus was in germinal

vesicle break down (GVBD), metaphase I (MI) or in metaphase II (MII) stages.

Maturation efficiency was calculated by MII/total oocytes cultured. Thereafter, oocytes

were also examined under a fluorescence microscope (Nikon, Eclipse 80i, Tokyo, Japan)

for evaluation of live/dead fluorescent staining. The emitted fluorescent signals of

calcein-AM and ethidium homodimer-1 were collected at 488 and 568 nm, respectively.

Oocytes were considered viable when the cytoplasm was stained positively with calcein-

AM (green) and chromatin was not labelled with ethidium homodimer-1 (red) and they

showed a normal chromatin configuration.

Fertilization parameters were evaluated at 18 hpi. The oocytes were considered

penetrated when they contained one or more swollen sperm heads and/or male pronuclei,

with their corresponding sperm tails, and two polar bodies. The fertilization parameters

evaluated were penetration rate (number of oocytes penetrated/total matured),

monospermy (number of oocytes containing only one male pronucleus/total penetrated),

number of spermatozoa/oocyte (mean number of spermatozoa in penetrated oocytes), and

efficiency of fertilization (number of monospermic oocytes/total inseminated).

At 2 and 7 days after IVF, the cleavage rate (number of oocytes divided to 2–4

cells/total) and blastocyst formation rate (number of blastocyst/total cleaved),

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respectively, were evaluated under a stereomicroscope. An embryo that had cleaved to

the two-cell stage or beyond was counted as cleaved, and an embryo with a clear

blastocele was defined as a blastocyst


Statistical analysis was conducted with Sigma Plot 11.0 (Systat Software Inc.,

USA). For percentage comparison between treatments, chi-square test and Fisher's exact

test were used. Data of means comparison among treatments of n° spermatozoa/oocyte

was performed by Mann-Whitney test. Data was presented as mean (± SEM) and

percentages. In all cases, a probability of P<0.05 indicated a significant difference.

5. Acknowledgements


authors thank Thalles Gothardo Pereira Nunes and Renato Felix da Silva, for assistance with

this study.

6. Conflicts of Interest




123

7. Figures and tables

Figure 1. Experimental design and endpoints of experiment 1 and 2.

In vitro maturation (IVM), in vitro fertilization (IVF), in vitro embryo culture (IVC),

hours post insemination (hpi).

124

Table 1. Rates of viable oocytes, germinal vesicle (GV), meiotic resumption and metaphase II (MII) rates, after in vitro maturation of

porcine oocytes in control medium alone or supplemented with DXR, A. oncocalyx or onco A (experiment 1).

Treatments Total Viable oocytes

Oocyte chromatin configuration Maturation

Efficiency

GV Meiotic

resumption* GVBD MI MII MII / total

(n) % % % % % % %

Control 157 81.53 (128/157) A 7.81 (10/128) A 92.19 (118/128) B 2.34 (3/128) 26.56 (34/128) B 63.28 (81/128) A 51.59 (81/157) A

DXR 187 12.83 (24/187) D 8.33 (2/24) A 91.67 (22/24) AB - 45.83 (11/24) AB 45.83 (11/24) AB 5.88 (11/187) C

A. oncocalyx 181 48.62 (88/181) C 7.95 (7/88) A 92.05 (81/88) B - 59.09 (52/88) A 32.95 (29/88) B 16.02 (29/181) B

Onco A 163 59.51 (97/163) B 1.03 (1/97) A 98.97 (96/97) A - 64.95 (63/97) A 34.02 (33/97) B 20.25 (33/163) B

A,B,C,D Distinct capital letters represent significant differences among treatments (P < 0.05).

n Total number of analyzed oocytes per treatment. * Includes GVBD, MI and MII oocytes.

125

Table 2. Rates of viable oocytes, matured, penetrated, monospermy and efficiency rates and number of spermatozoa per oocyte after

previous exposure (only during in vitro maturation) to DXR, A. oncocalyx or onco A(experiment 1).

Endpoints *

Treatments Total Viability Matured / viable

Penetrated /

matured

Monospermy /

penetrated

IVF Efficiency

(2pn/total) # SPZ / oocyte

(n) % % % % % %

Control 251 78.49 (197/251) A 96.95 (191/197) A 67.02 (128/191) A 57.03 (73/128) A 29.08 (73/251) A 1.94+ 0.14 A

DXR 170 38.82 (66/170) C 93.93 (62/66) A 77.42 (48/62) A 52.08 (25/48) A 14.71 (25/170) B 1.72+ 0.17 A

A. oncocalyx 291 68.73 (200/291) B 77.50 (155/200) B 76.77 (119/155) A 45.38 (54/119) A 18.56 (54/291) B 2.15+ 0.13 A

Onco A 252 66.67 (168/252) B 83.92 (141/168) B 69.50 (98/141) A 51.02 (50/98) A 19.84 (50/252) B 2.15 + 0.2 A A,B,C Distinct capital letters represent significant differences among treatments (P < 0.05).

n Total number of analyzed oocytes per treatment. * All the endpoints were evaluated 18 hpi.

126

Figure 2. Percentage of cleaved (A) and blastocyst/cleaved (B) after previous exposure (only during in vitro maturation) to DXR, A.

oncocalyx or onco A, only.a,b,c Distinct letters represent significant differences among treatments (P < 0.05) (experiment 1).

127

Table 3. Rates of viable oocytes, matured, penetrated, monospermy and efficiency rates and number of spermatozoa per oocyte after 18hpi

exposure (only during in vitro embryo culture) to DXR, A. oncocalyx or onco A (experiment 2).

Endpoints *

Treatments Total Viability Matured / viable

Penetrated /

matured

Monospermy /

penetrated

Efficiency

(2pn/total) # SPZ / oocyte

(n) % % % _% % %

Control 71 71.83 (51/71) AB 96.07 (49/51) A 85.71(42/49) A 54.76 (23/42) A 32.39 (23/71) A 1.43 + 0.1 B

DXR 87 59.7 (52/87) B 98.07 (51/52) A 64.7 (33/51) B 45.45 (15/33) AB 17.24 (15/87) BC 1.86+ 0.16 AB

A. oncocalyx 88 75 (66/88) A 93.93 (62/66) A 69.35 (43/62) B 51.16 (22/43) AB 25 (22/88) AC 1.72+ 0.14 B

Onco A 73 58.9 (43/73) B 97.67 (42/43) A 59.52 (25/42) B 28 (7/25) B 9.59 (7/73) B 2.51 + 0.28 A

A,B,C Distinct capital letters represent significant differences among treatments (P < 0.05).

n Total number of analyzed oocytes per treatment. * All the endpoints were evaluated 18 hpi.

128

Figure 3. Percentage of cleaved (A) and blastocyst / cleaved (B) after exposure (only during in vitro embryo culture) to DXR, A. oncocalyx

or onco A. a,b Distinct letters represent significant differences among treatments (P < 0.05) (experiment 2).

129

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133

10 CONCLUSÕES

Os resultados obtidos ao longo desta tese permitiram concluir que:

Fase I:

A. oncocalyx e onco A afetaram a foliculogênese caprina in vitro de uma forma

concentração-dependente, apresentando-se menos tóxica do que a DXR.

Fase II:

A. oncocalyx e onco A não apresentaram efeito tóxico sobre o desenvolvimento

de folículos secundários isolados caprinos, nem sobre as taxas de maturação in

vitro de CCOs. No entanto, estas drogas influenciaram negativamente a

viabilidade dos oócitos caprinos após a MIV.

A DXR, ao contrário da A. oncocalyx e onco A, reduziu a sobrevivência, formação

de antro e crescimento folicular de folículos pré-antrais isolados caprinos

cultivados in vitro.

Fase III:

A adição de DXR durante a MIV ou o CIV afetou negativamente a eficiência da

FIV e as taxas de clivagem na espécie suína. Além disso, a exposição de CCOs à

DXR apenas durante MIV, foi mais prejudicial à viabilidade oocitária e a

formação de blastocistos, do que a A. oncocalyx e a onco A.

134

11 PERSPECTIVAS

A tecnologia do ovário artificial foi utilizada com sucesso no presente trabalho

para a realização de um teste de toxidade reprodutiva comparando-se uma droga utilizada

comercialmente (DXR) para a terapia do câncer, com substâncias (A. oncocalyx e onco

A) oriundas de uma planta (Pau-Branco do sertão) pertencentemente ao bioma da

caatinga, com potencial anticancerígeno. O efeito menos tóxico da onco A em relação a

DXR sobre a foliculogênese e o desenvolvimento embrionário, abre novas perspectivas

para o emprego da referida substância no tratamento de câncer em mulheres. No entanto,

mais testes são necessários para um melhor entendimento da atuação das substâncias e

seus efeitos sobre a reprodução.

135

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